Production of insulin producing cells

ABSTRACT

A population of enteroendocrine cells (EEC) is obtained from a mammalian post-natal cell population, such as a population including post-natal stem cells, by treating the population with a plurality of small molecules that upregulate ChgA and promote differentiation of the cells to form the enteroendocrine cells. The upregulation of ChgA is such that the fraction of cells expressing CGA in the obtained cell population, as measured by a ChgA Immunostaining Assay, is at least about 1.5%. Small molecules that can be used to differentiate the post-natal cells into the enteroendocrine cells can include at least one of a Wnt activator, a Notch inhibitor, a Wnt inhibitor, a MEK/ERK inhibitor, a growth factor, a HDAC inhibitor, a Histone Methylation Inhibitor, a Tgf-β inhibitor, and a NeuroD1 activator. Also, the insulin expression of a population of mammalian cells is increased by treating the population with a plurality of small molecules that increase the insulin expression.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/400,877, filed Jan. 6, 2017, which claims the benefit of U.S.Provisional Application No. 62/276,814, filed on Jan. 8, 2016. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. R01DE013023 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND

The enteroendocrine system orchestrates how the body responds tonutrients by employing a diversity of hormones to fine-tune a wide rangeof physiological responses in the body, thus playing an important rolein digestive and metabolic diseases such as Gastrointestinal (GI)disorders, diabetes and obesity. Enteroendocrine cells (EECs) form thelargest endocrine system in the body (Grible and Reimann, 2015). EECsare individually dispersed along the crypt-villus axis throughout theintestinal epithelial but only exist in a small percentage (˜1%) in vivo(Gunawardene et al., 2011). A key function of EECs is to sense luminalcontents, particularly nutrients, and to respond by the secretion of adiversity of hormones (e.g. GLP-1) which modulate food intake, energyhomeostasis and glucose tolerance (Furness, 2013). It is also suggestedthat EECs play a key role in gastric bypass surgery by secretinghormones such as GLP-1, PYY and GLP-2 (Mumphrey, 2013). Accordingly,EECs are a therapeutic target in diabetes and obesity. Furthermore,mounting evidence demonstrates an immunoregulatory function of EECs ininnate immunity (Moran, 2008). EECs express functional Toll-likereceptors (TLR) and directly respond to metabolites produced bycommensal bacteria (Bogunovic, 2007). Recent evidence also suggests thatEECs may directly orchestrate immune cell function through alteration innumber and hormone secretion during inflammation (Worthington, 2015).EECs are also believed to play a critical role in metabolic diseases(e.g. diabetes and obesity), and gastrointestinal pathologies such asirritable Bowel syndrome, infectious enteritis and inflammatory boweldisease (Moran, 2008 and Manocha and Kahn, 2012). Thus, there is greatinterest in EECs for the exploration and development of diseaseinterventions.

However, the study of enteroendocrine cells has been hindered by therelative lack of ability to culture EECs in vitro, as well as by thedispersed distribution of EEC and relative scarcity (1%) of EECs inintestinal epithelium. In particular, the knowledge of signals thatcontrol the differentiation and function of EEC are largely unknown.Furthermore, direct in vitro study of EECs has not been possible,because they are terminally differentiated cells that do not divide.Thus, due to the dispersed distribution and scarcity (1%) of EECs in thegut epithelium, it has been difficult to study the function andregulation of EECs in situ (Sternini, Anselmi and Rosengut, 2008 andGunawerdene, Corfe and Staton, 2011).

Accordingly, there remains a need for in vitro EEC culture to allow forinvestigation of metabolic and digestive diseases. There is also a needfor the ability to modulate the function of EECs and obtain specific EECsub-types, such as for use in the discovery and implementation of thetreatment of disease states.

SUMMARY OF THE INVENTION

Aspects of the disclosure include obtaining a population ofenteroendocrine cells (EECs) from a mammalian post-natal cellpopulation, such as a post-natal stem cell population, by treating thepopulation with a plurality of small molecules that upregulate ChgA andpromote differentiation of the cells to form the enteroendocrine cells.The upregulation of ChgA is such that the fraction of cells expressingCGA in the obtained cell population, as measured by a ChgAImmunostaining Assay, is at least about 1.5%. Small molecules that canbe used to differentiate the post-natal stem cells into theenteroendocrine cells can include at least one of a Wnt activator, aNotch inhibitor, a Wnt inhibitor, a MEK/ERK inhibitor, a growth factor,a HDAC inhibitor, a Histone Methylation Inhibitor, a Tgf-β inhibitor,and a NeuroD1 activator.

Aspects of the disclosure also include a method for increasing theinsulin expression of a population of mammalian cells by treating thepopulation with a plurality of small molecules that induce the cells toincrease the insulin expression. The insulin expression level may beincreased in the cell population such that, in an Insulin Activity Assayusing an Insulin-GFP reporter, the fraction of cells having the InsulinGFP reporter activated is at least about 1%. The mammalian cells thatcan be treated to increase the insulin expression can include post-natalcells or enteroendocrine cells, such as post-natal stem cells,post-natal multipotent progeny cells, and enteroendocrine cells. Smallmolecules that can be used for the treatment to increase the expressionof insulin in the cells can include a DNA methylation inhibitor, a Tgf-βinhibitor, and a NeuroD1 activator.

Methods and/or compositions for treating disease states with the EECsand/or insulin producing cells described herein are also includedaccording to an aspect of the disclosure. Also, populations of cellscorresponding to the obtained EECS and/or insulin producing cells arealso included according to an aspect of the disclosure, as are kitscontaining the small molecules for use in preparing the EEC and/orinsulin producing cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIG. 1 shows an increase in differentiation of enteroendocrine cells(EEC) from ISCs using a Notch inhibitor.

FIG. 2 shows mRNA expression of ChgA in cultured intestinal stem cells.

FIG. 3 shows a 96-well screening platform for small molecules thatincrease EEC differentiation.

FIG. 4 shows positive hits from small molecules screening results.

FIG. 5 shows validation of the positive hits using small molecules andintestinal stem cells differentiated under END conditions.

FIG. 6 shows MAPK/ERK or EGFR inhibitors specifically increase EECdifferentiation.

FIG. 7 shows small molecules Gefitinib (Ge), AS703026 (As) or PD0325901(Pd) decrease Ngn3 expression during EEC differentiation, and thatR-Spondin 1 promotes Ngn3 expression.

FIG. 8 shows an optimized differentiation protocol.

FIG. 9 shows Ngn3 expression at 24 h during EEC differentiation.

FIG. 10 shows expression of key markers of EEC during differentiation atday 3.

FIG. 11 shows a time course study of key genes during EECdifferentiation.

FIG. 12 shows a 2-step protocol with increased EEC marker ChgAexpression after 5 days differentiation.

FIG. 13 shows a 2-step protocol with increased EEC differentiation bystaining EEC marker ChgA following 5 days differentiation. Scale bar: 50μm.

FIG. 14 shows improvement of the differentiation protocol by additionalsmall molecules.

FIG. 15 shows Tubastatin A (Tu) increases EEC differentiation when addedin Step 1 of the differentiation protocol.

FIG. 16 shows Tranylcypromine increased EEC differentiation when addedat both steps in differentiation.

FIG. 17 shows that removal of EGF further increased differentiation ofEEC.

FIG. 18 shows an improved differentiation protocol.

FIG. 19 shows highly efficient EEC differentiation from ISC. Scale bars:20 μm.

FIG. 20 shows functional L cells (GLP-1) generated from ISC.

FIG. 21 shows additional factors, including Wnt-059 and ISX-9 and theircombination, increase EEC differentiation.

FIG. 22 shows expression of functional EEC markers under multipleconditions.

FIG. 23 shows a differentiation protocol for EEC differentiation fromISC.

FIG. 24 shows the combination of 5-Aza (5), 616452 (6), and Wnt-059 (C)induce Insulin-GFP expression in differentiated intestinal stem cells atday 5.

FIG. 25 shows a dose response of 5-Aza in inducing insulin-GFPexpression at day 5.

FIG. 26 shows a dose response of 616452 in inducing insulin-GFPexpression at day 5.

FIG. 27 shows that ISX-9 (I) further increased insulin-GFP expressionafter 5 days in culture.

FIG. 28 shows a dose response of ISX-9 in inducing insulin-GFPexpression at day 5.

FIG. 29 shows a FACS analysis of Insulin-GFP expression of cells inmultiple conditions after 5 days in culture.

FIG. 30 shows GFP and brightfield of cells treated without or with drugsat day 7.

FIG. 31 shows gene expression data.

FIG. 32 shows treatment of cells with low (2 mM) and high (20 mM)concentration of glucose induce different levels of insulin release.

FIG. 33 shows a flow diagram of a differentiation protocol.

FIG. 34 shows a model of in vivo EEC differentiation controlled by Wntand Notch pathways.

FIG. 35 shows that a combination of Notch and EGFR/MEK/ERK inhibitionand Wnt inactivation (by R-Spondin1 withdraw) induces specification ofISCs towards EEC direction as determined by immunostaining against ChgA.

FIG. 36 shows that removing EGF and/or Noggin from the combinationinduced higher level ChgA expression, while adding the Wnt pathwayinhibitor IWP 2 (or I) or GSK3β inhibitor CHIR99021 (or C) did notfurther increase differentiation towards EEC.

FIG. 37 shows the morphology of cell colonies in multipledifferentiation conditions.

FIG. 38 shows the morphology of cell colonies in multipledifferentiation conditions. Note the prominent lumen of colonies withdead cells in conditions of Notch, MEK inhibition and Wnt inactivation(D.Pd condition) as well as Notch/MEK/Wnt inhibition (D.Pd.C59condition). While 2-step differentiation condition (ENRD-END.Pd) inducedless cell death.

FIGS. 39A-39E show that additional small molecules increase EECdifferentiation. FIG. 39A. mRNA expression of Gip, Gcg, Cck for cellsdifferentiated in multiple conditions, with or without the addition ofTubastatin A (Tu) in step 1. FIG. 39B. mRNA expression of Gip, Gcg andCck for cells differentiation in multiple conditions with or withoutTranylcypromine (Tc) added in Step 1, Step 2 or both steps. FIG. 39C.Dose dependent induction of EEC markers (ChgA, Gcg, Gip, Tph1) forTranylcypromine. FIG. 39D. mRNA expression of multiple EEC markers (Gip,Gcg, Cck) for cells differentiated in multiple conditions. FIG. 39E.mRNA expression of Lyz1 for cells differentiated in multiple conditions.

FIGS. 40A-40E show that protocol optimization results in high efficientdifferentiation of EECs from ISCs. FIG. 40A. mRNA expression of markersfor goblet cell (Muc2), Paneth cell (Lyz1) and EECs (ChgA, Gcg, Gip,Cck, Tph1, Sst, Sct, and Pyy) with or without the addition of Wntpathway inhibitor Wnt-059 (C59) and Tgf-β pathway inhibitor Repsox (Rep)at multiple time points. S1, S2 represent Step 1 and Step 2 of 2-stepdifferentiation protocol. 0 h or 12 h indicates the time points ofadding C59. ENR was used as control for spontaneous differentiation.FIG. 40B. Time-course study of multiple genes in 2-step differentiationprocess. FIG. 40C. expression level of multiple genes comparing withtheir corresponding peak levels (Sox9, Math1, Ngn3, and NeuroD1 at day2, ChgA at day 5, see FIG. 40D). FIG. 40E. Immunostaining ofdifferentiated EEC cells.

FIGS. 41A-41C show that protocol optimization results in high efficientdifferentiation of EECs from ISCs. FIG. 41A. mRNA expression of multiplegenes in conditions with or without Repsox. Basal condition used wasENR.D.Tu.Tc-D.Pd.Tc.C59. Repsox was added in Step 2. FIG. 41B. mRNAexpression of multiple genes in conditions as indicated. FIG. 41C.Morphology of differentiated cell colonies. Arrows indicate dead cellsexpelled from the colonies.

FIG. 42 shows that RepSox and FGF10 increased Insulin mRNA expressionwhen added in Stage 1.

FIG. 43 shows that longer 5Aza treatment increased Insulin mRNAexpression.

FIG. 44 shows that delayed addition of Wnt-059 in Stage 2 increasedInsulin mRNA expression.

FIG. 45 shows that BayK 8644 increased Insulin mRNA expression whenadded in Stage 1.

FIG. 46 shows dose-dependent activity of BayK 8644 in promoting Insulinexpression.

FIG. 47 shows that BayK 8644 increased Insulin mRNA expression whenadded in Stages 1-3.

FIG. 48 shows dose-dependent activity of DAPT in promoting Insulinexpression.

FIG. 49 shows that IOX1 increased insulin and Nkx6.1 expression.

FIG. 50. shows a dose response of RepSox in promoting insulin and Nkx6.1expression.

FIG. 51 shows a dose response of ISX9 in promoting insulin and Nkx6.1expression.

FIG. 52 shows a dose response of Wnt-059 in promoting insulin and Nkx6.1expression.

FIG. 53 shows that DMH-1 increased insulin and Nkx6.1 expression levelwhen added in Stage 2-3.

FIG. 54 shows that Dexamethasone increased insulin and Nkx6.1 expressionlevel when added in Stage 2-3.

FIG. 55 shows that T3 increased Insulin mRNA expression when added inStage 3.

FIG. 56 shows that T3 and N-acetylcysteine (N-Alc) increased InsulinmRNA expression when added in Stage 3.

FIG. 57 shows that CHIR99021 (CHIR) increased Insulin mRNA expressionwhen added in Stage 3.

FIG. 58 shows that Exendin-4 and Aurora Kinase Inhibitor II, as well asForskolin increased Insulin mRNA expression.

FIG. 59. shows that GC1 can replace T3 in promoting Insulin mRNAexpression.

FIG. 60 shows Insulin-GFP expression and morphology of islet-likestructures obtained using combination of factors, from gastrointestinalstem cells.

FIG. 61 shows a diagram of a cell culture process.

DEFINITIONS

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration” refers to introducing a substance into a subject. Insome embodiments, administration is oral, or by injection. In certainembodiments “causing to be administered” refers to administration of asecond component after a first component has already been administered(e.g., at a different time and/or by a different actor).

An “antibody” refers to an immunoglobulin polypeptide, or fragmentthereof, having immunogen binding ability.

As used herein, an “agonist” is an agent that causes an increase in theexpression or activity of a target gene, protein, or a pathway,respectively. Therefore, an agonist can bind to and activate its cognatereceptor in some fashion, which directly or indirectly brings about thisphysiological effect on the target gene or protein. An agonist can alsoincrease the activity of a pathway through modulating the activity ofpathway components, for example, through inhibiting the activity ofnegative regulators of a pathway. Therefore, a “Wnt agonist” can bedefined as an agent that increases the activity of Wnt pathway, whichcan be measured by increased TCF/LEF-mediated transcription in a cell.Therefore, a “Wnt agonist” can be a true Wnt agonist that bind andactivate a Frizzled receptor family member, including any and all of theWnt family proteins, an inhibitor of intracellular beta-catenindegradation, and activators of TCF/LEF. A “Notch agonist” can be definedas an agent that increase the activity of Notch pathway, which can bedetermined by measuring the transcriptional activity of Notch.

An “antagonist” refers to an agent that binds to a receptor, and whichin turn decreases or eliminates binding by other molecules.

“Cell Density” as used herein in connection with a specific cell type isthe mean number of that cell type per area in a RepresentativeMicroscopy Sample. The cell types may include but are not limited toLgr5⁺ cells, enteroendocrine cells, or insulin producing cells. The CellDensity may be assessed with a given cell type in a given sample, organor tissue.

“Cell Differentiation” refers to the process by which a cell becomesspecialized to perform a specific function, such as in the conversion ofpost-natal stem cells into cells having a more specialized function. Inan embodiment, Lgr5+ intestinal stem cells are differentiated intoenteroendocrine cells.

“ChgA Immunostaining Assay” as used herein is an assay used to determinethe fraction of cells in a cell population that express ChgA by animmunostaining method. In an example of a ChgA immunostaining assay, acell culture medium in which a cell population has been treated isremoved, and the sample is washed with PBS. Organoids or cell coloniescultured in Matrigel are fixed directly by adding 4% PFA and incubatingfor 20 mins at room temperature. The Matrigel is then mechanicallydisrupted, and the cells are transferred to BSA-coated Eppendorf tubes.Samples are washed with PBS, permeabilized with 0.25% Triton X-100 for30 minutes, and stained with primary antibody against Chromogranin A(e.g. anti-chromogranin A, sc-13090, Santa Cruz) and appropriatesecondary antibodies (e.g. Alexa Fluor conjugated secondary antibodies,such as Alexa Fluor 594 conjugated Donkey anti-Rabbit antibody, A-21207;Life Technologies). Images are acquired by confocal microscopy.

“CHIR99021” is a chemical composition having the chemical formulaC₂₂H₁₈Cl₂N₈ and the following alternate names: CT 99021;6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile.Its chemical structure is as follows:

“Complementary nucleic acid sequence” refers to a nucleic acid sequencecapable of hybridizing with another nucleic acid sequence comprised ofcomplementary nucleotide base pairs.

“Cross-Sectional Cell Density” as used herein in connection with aspecific cell type is the mean number of that cell type per area ofcross section through a tissue in a Representative Microscopy Sample.

“Decreasing” and “decreases” refer to decreasing by at least 5%, forexample, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99 or 100%, for example, as compared to thelevel of reference, and includes decreases by at least 1-fold, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 500, 1000-fold or more, for example, as compared to thelevel of a reference.

“EGFR inhibitor” is substance that inhibits the epidermal growth factorreceptor. Examples of EGFR inhibitors include Erlotinib HCl (OSI-744),Gefitinib (ZD1839), Lapatinib (GW-572016) Ditosylate, Afatinib(BIBW2992), Neratinib (HKI-272), Canertinib (CI-1033), Lapatinib, AG-490(Tyrphostin B42), CP-724714, Dacomitinib (PF299804, PF299), WZ4002,AZD8931 (Sapitinib), CUDC-101, AG-1478 (Tyrphostin AG-1478), PD153035HCl, Pelitinib (EKB-569), AC480 (BMS-599626), AEE788 (NVP-AEE788),OSI-420, WZ3146 WZ8040, AST-1306, Rociletinib (CO-1686, AVL-301),Genistein, Varlitinib, Icotinib, TAK-285, WHI-P154, PD168393, CNX-2006,Tyrphostin 9, AG-18, Poziotinib (HM781-36B), AZD3759, Osimertinib(AZD9291), Afatinib (BIBW2992) Dimaleate, Erlotinib, Olmutinib (HM61713,BI 1482694), CL-387785 (EKI-785), NSC228155, AZ5104, AG490, AG 494, AG555, AG 556, AG 825, AG 879, AG 99, AP 24534, AV 412, BIBU 1361, BIBX1382, BMS 599626, Canertinib, CGP 52411, GW 583340, HDS 029, HKI 357,Iressa, JNJ 28871063, Lavendustin A, Methyl 2,5-dihydroxycinnamate, PD158780, PF 6274484, PKI 166, R 1530, RAF 265, and XL 184, among others.

“Eliminate” means to decrease to a level that is undetectable.

“Enteroendocrine cells” refers to cells that are specialized endocrinecells of the gastrointestinal tract and pancreas, and can be found inthe intestinal tract, stomach and pancreas. The enteroendocrine cellsform the largest endocrine system in the body, and can sense luminalcontents, particularly nutrients, and respond by the secretion of adiversity of hormones (e.g. GLP-1) which modulate food intake, energyhomeostasis and glucose tolerance. Specific types of enteroendocrinecell are often classified according to the expression of hormones withinthe specific enteroendocrine cell subset, such as cells that expressGLP-1, SHT, SST, gastrin, CCK, SCT, NTS, PYY, Gastrin and Ghrelin, amongothers. The different subsets of enteroendocrine have also beensometimes referred to as K cells, I cells, L cells, G cells,Enterochromaffin cells, N cells and S cells, but increasingly thehormone expression of the cells is used to identify the cell subtypes,as set forth above. Enteroendocrine cells can be identified byexpression of ChgA marker, which can be detected by assays such as themRNA ChgA Expression Assay and ChgA Immunostaining Assay describedherein.

“Engraft” or “engraftment” refers to the process of stem or progenitorcell incorporation into a tissue of interest in vivo through contactwith existing cells of the tissue.

“Epithelial progenitor cell” refers to a multipotent cell which has thepotential to become restricted to cell lineages resulting in epithelialcells.

“Epithelial stem cell” refers to a multipotent cell which has thepotential to become committed to multiple cell lineages, including celllineages resulting in epithelial cells.

“Fragment” refers to a portion, e.g., of a polypeptide or nucleic acidmolecule. This portion contains, for example, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the referencenucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30,40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900,or 1000 nucleotides or amino acids.

“Growth factor” refers to a substance capable of stimulating cellulargrowth, proliferation or differentiation.

“GSK3beta,” “GSK3β,” and “GSK3B” as used interchangeably herein areacronyms for glycogen synthase kinase 3 beta.

“GSK3beta inhibitor” is a substance that inhibits the activity ofGSK3beta.

“HDAC” as used herein is an acronym for histone deacetylase.

“HDAC inhibitor” is a substance that inhibits the activity of HDAC.

“Histone Methylation Inhibitor” is a substance that inhibits histonemethylation.

“Hybridize” refers to pairing to form a double-stranded molecule betweencomplementary nucleotide bases (e.g., adenine (A) forms a base pair withthymine (T), as does guanine (G) with cytosine (C) in DNA) undersuitable conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) MethodsEnzymol. 152:507).

An “inhibitor” refers to an agent that causes a decrease in theexpression or activity, e.g., of a target gene, a protein, or a pathway.For example, an “Wnt inhibitor” refers an agent that causes a decreasein the activity of Wnt signaling pathway, which can be for example a Wntreceptor inhibitor, a Wnt receptor antagonist, a Porcupine inhibitorwhich inhibits Wnt secretion, or a Tankyrase inhibitor, or a drug thatinterferes with β-catenin interactions. An “antagonist” can be aninhibitor, but is more specifically an agent that binds to a receptor,and which in turn decreases or eliminates binding by other molecules.

“Increasing” and “increases” refer to increasing by at least 5%, forexample, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 99, 100% or more, for example, as compared tothe level of a reference, and includes increases by at least 1-fold, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, 200, 500, 1000-fold or more, for example, as compared to thelevel of a as compared to the level of a reference standard.

“Insulin Activity Assay” as used herein is an assay used to determinethe extent to which insulin gene transcription, translation or insulinrelease has been activated in a cell population. In an exemplary InsulinActivity Assay, the initial cells are isolated from the intestine of anInsulin-GFP mouse such as B6.Cg-Tg (Ins1-EGFP)1Hara/J mouse (alsoreferred to as the MIT-GFP mouse, Jackson lab stock no.: 006864).Intestinal crypts are isolated from the proximal half of the intestine.Approximately 200 crypts are entrapped in 40 μl of Matrigel and culturedin 24-well plates with 500 μl of crypt culture media (Advanced DMEM/F12with media Supplements (1X N2, 1X B27, 2 mM Glutamax, 10 mM HEPES, 1 mMN-acetylcysteine, and 100 U/ml Penicillin/100 μg/ml Streptomycin)), andsupplemented with growth factors (50 ng/ml EGF, 100 ng/ml Noggin, and500 ng/ml R-Spondin1) and small molecules (5 μM CHIR99021 and 1.25 mMVPA) to obtain an enriched population of intestinal stem cells. Thecells are then passaged for 1-2 times to create a starting cellpopulation for the assay. To test the capacity of agents to activateinsulin gene transcription, translation or insulin release, the cellsare incubated with appropriate culture media (e.g. aforementioned cryptculture media), growth factors and/or other agents being tested.Appropriate culture media, including crypt culture media as well as theagents being assessed, are added into each well and incubated with thecells for a period of 2-10 days with media change every 2 days. Thefraction of insulin-GFP positive cells (i.e., cells in which theinsulin-GFP reporter is activated) can be quantified using a flowcytometer to measure the fraction of GFP+ cell population present in thetotal cell population. Also, the average insulin activity of a cellpopulation can be measured by measuring the average mRNA expressionlevel of insulin of the population normalized using suitable referencesor housekeeping genes (e.g., using mRNA expression of Hprt as abaseline).

“Intestinal stem cell” refers to a multipotent cell of intestinallineage which has the potential to become committed to multiple celllineages, including cell lineages resulting in intestinal cells such asenteroendocrine cells, enterocyte cells, goblet cells and paneth cells.

“Isolated” refers to a material that is free to varying degrees fromcomponents which normally accompany it as found in its native state.“Isolate” denotes a degree of separation from original source orsurroundings.

“Lgr5” is an acronym for the Leucine-rich repeat-containing G-proteincoupled receptor 5, also known as G-protein coupled receptor 49 (GPR49)or G-protein coupled receptor 67 (GPR67). It is a protein that in humansis encoded by the Lgr5 gene.

“Lgr5+ cell” or “Lgr5-positive cell” as used herein is a cell thatexpresses Lgr5. “Lgr5− cell” as used herein is a cell that is not Lgr5+.

“Mammal” refers to any mammal including but not limited to human, mouse,rat, sheep, monkey, goat, rabbit, hamster, horse, cow or pig.

“Multipotent progeny cell” refers to refers to a cell that is alreadymore specific than a stem cell, meaning it has the tendency todifferentiate into a specific type of cell, but still retains theability to differentiate into multiple different but limited cell types.The multipotent progeny cell may be a multipotent cell that has beendifferentiated from a stem cell but has not yet differentiated into a“target” cell type.

“MEK/ERK inhibitor” is a substance that inhibits the MEK/ERK signalingpathway. Examples of MEK inhibitors include Arctigenin, BIX 02189,10Z-Hymenialdisine, PD 0325901, PD 184352, PD 198306, PD 334581, PD98059, SL 327, U0126, Selumetinib (AZD6244), Trametinib (GSK1120212),PD184352 (CI-1040), PD98059, Pimasertib (AS-703026), BIX 02188, TAK-733,AZD8330, Binimetinib (MEK162, ARRY-162, ARRY-438162), PD318088,Refametinib (RDEA119, Bay 86-9766), BI-847325, Cobimetinib (GDC-0973,RG7420), GDC-0623, and APS-2-79. Examples of ERK inhibitors includeSCH772984, DEL-22379, VX-11e, ERKS-IN-1, XMD8-92, SC1 (Pluripotin),Ulixertinib (BVD-523, VRT75227, FR 180204, GDC-0994, BIX 02189, TCS ERK11e, TMCB, and Eicosapentaenoic Acid.

“mRNA ChgA Expression Assay” refers to an assay used to determine therelative ChgA mRNA expression level in a cell population. For example,the assay can determine ChgA mRNA expression level of a differentiatedcell population following treatment with agents being tested, ascompared to an untested population of post-natal stem cells. In anexample of an mRNA ChgA Expression Assay, the cells were collected andthe RNA is extracted from the cells using an RNA extraction kit (such asRNeasy Mini kit, Qiagen). The ChgA expression level is then assessed byQuantitative real-time PCR using a one-step qPCR kit (such as QuantiTechProbe PCR kit, Qiagen) and ChgA primers and probes (such as commerciallyavailable Taqman probe for mouse ChgA, Life Technologies).

“mRNA Insulin Expression Assay” refers to an assay used to determine therelative insulin mRNA expression level of a cell population. Forexample, the assay can determine the insulin mRNA level expressed in acell population treated to increase the expression of insulin in thecells, as compared to an untreated population of cells. In an exemplarymRNA Insulin Expression Assay, the cells were collected and the RNA isextracted from the cells using an RNA extraction kit (such as RNeasyMini kit, Qiagen). The insulin expression level is then assessed byQuantitative real-time PCR using a one-step qPCR kit (such as QuantiTechProbe PCR kit, Qiagen) and Ins1 or Ins2 primers and probes (such as thecommercially available TaqMan probe for mouse Ins1 and Ins2, LifeTechnologies). The relative insulin mRNA expression can be determinedrelative to a baseline or other marker, such as Hprt mRNA expressionlevel or other baseline marker.

“NeuroD1 activator” is a substance that activates NeuroD1.

“Non-human mammal”, as used herein, refers to any mammal that is not ahuman.

“Notch inhibitor” refers to an inhibitor of the Notch signaling pathway.

“Ngn3” refers to Neurogenin-3, a protein expressed in endocrineprogenitor cells and associated with enteroendocrine differentiation.

“Ngn3 Assay” as used herein is an assay used to determine the extent ofexpression of Ngn3 in cells, such as cells subjected to adifferentiation protocol. In an exemplary Ngn3 Assay, RNA is isolatedfrom cells that have been cultured according to a differentiationprotocol, and quantitative real-time PCR is performed using commerciallyavailable primers and probes (such as Taqman probes) to determine anextent of Ngn3 expression in the cells.

As used in relevant context herein, the term “number” of cells can be 0,1, or more cells.

“Organoid” or “epithelial organoid” refers to a cell cluster oraggregate that resembles an organ, or part of an organ, and possessescell types relevant to that particular organ.

“Population” of cells refers to any number of cells greater than 1, andeven at least 1×10³ cells, at least 1×10⁴ cells, at least at least 1×10⁵cells, at least 1×10⁶ cells, at least 1×10⁷ cells, at least 1×10⁸ cells,at least 1×10⁹ cells, or at least 1×10¹⁰ cells.

“Post-natal cell” refers to a non-embryonic cell. Post-natal cells caninclude at least one of post-natal stem cells, post-natal progenitorcells, and post-natal multipotent progeny cells, as well as one or morecells differentiated from these cells, such as enteroendocrine cells(EECs).

“Post-natal stem cell” refers to non-embryonic stem cells that have thecapacity to self renew and to differentiate into multiple cell lineages.Post-natal stem cells may also be referred to as adult stem cells orsomatic stem cells. Post-natal stem cells can include intestinal stemcells, epithelial stem cells, hematopoietic stem cells, mammary stemcells, mesenchymal stem cells, endothelial stem cells and neural stemcells.

“Progenitor cell” as used herein refers to a cell that, like a stemcell, has the tendency to differentiate into a specific type of cell,but is already more specific than a stem cell and is pushed todifferentiate into its “target” cell.

“Reference” means a standard or control condition (e.g., untreated witha test agent or combination of test agents).

The term “sample” refers to a volume or mass obtained, provided, and/orsubjected to analysis. In some embodiments, a sample is or comprises atissue sample, cell sample, a fluid sample, and the like. In someembodiments, a sample is taken from (or is) a subject (e.g., a human oranimal subject). In some embodiments, a tissue sample is or comprisesbrain, hair (including roots), buccal swabs, blood, saliva, semen,muscle, or from any internal organs, or cancer, precancerous, or tumorcells associated with any one of these. A fluid may be, but is notlimited to, urine, blood, ascites, pleural fluid, spinal fluid, and thelike. A body tissue can include, but is not limited to, brain, skin,muscle, endometrial, uterine, and cervical tissue or cancer,precancerous, or tumor cells associated with any one of these.

“Self-renewal” refers to the process by which a stem cell divides togenerate one (asymmetric division) or two (symmetric division) daughtercells with development potentials that are indistinguishable from thoseof the mother cell. Self-renewal involves both proliferation and themaintenance of an undifferentiated state.

“Small molecule” as referred to herein refers to an organic compoundthat can participate in regulating biological pathways, and is anon-nucleic acid, is typically non-peptidic and non-oligomeric, and mayhave a molecular weight of less than 1500 daltons.

“Stem cell” refers to a multipotent cell having the capacity to selfrenew and to differentiate into multiple cell lineages.

“Stem Cell Markers” as used herein can be defined as gene products (e.g.protein, RNA, etc) that are specifically expressed in stem cells. Onetype of stem cell marker is gene products that directly and specificallysupport the maintenance of stem cell identity. Examples include Lgr5 andSox2. Additional stem cell markers can be identified using assays thatwere described in the literature. To determine whether a gene isrequired for maintenance of stem cell identity, gain-of-function andloss-of-function studies can be used. In gain-of-function studies, overexpression of specific gene product (the stem cell marker) would helpmaintain the stem cell identity. While in loss-of-function studies,removal of the stem cell marker would cause loss of the stem cellidentity or induced the differentiation of stem cells. Another type ofstem cell marker is a gene that is only expressed in stem cells but doesnot necessarily have a specific function to maintain the identity ofstem cells. This type of marker can be identified by comparing the geneexpression signature of sorted stem cells and non-stem cells by assayssuch as micro-array and qPCR. This type of stem cell marker can be foundin the literature (e.g. Liu Q. et al., Int J Biochem Cell Biol. 2015March; 60:99-111. http://www.ncbi.nlm.nih.gov/pubmed/25582750).Potential stem cell markers include Ccdc121, Gdf10, Opcm1, Phex, etc.The expression of stem cell markers such as Lgr5 or Sox2 in a given cellor cell population can be measured using assays such as qPCR,immunohistochemistry, western blot, and RNA hybridization. Theexpression of stem cell markers can also be measured using transgeniccells expressing reporters which can indicate the expression of thegiven stem cell markers, e.g. Lgr5-GFP or Sox2-GFP. Flow cytometryanalysis can then be used to measure the activity of reporterexpression. Fluorescence microscopy can also be used to directlyvisualize the expression of reporters. The expression of stem cellmarkers may further be determined using microarray analysis for globalgene expression profile analysis. The gene expression profile of a givencell population or purified cell population can be compared with thegene expression profile of the stem cell to determine similarity betweenthe 2 cell populations. Stem cell function can be measured by colonyforming assay or sphere forming assay, self-renewal assay anddifferentiation assay. In a colony (or sphere) forming assay, whencultured in appropriate culture media, the stem cell should be able toform colonies, on cell culture surface (e.g. cell culture dish) orembedded in cell culture substrate (e.g. Matrigel) or be able to formspheres when cultured in suspension. In a colony/sphere forming assay,single stem cells are seeded at low cell density in appropriate culturemedia and allowed to proliferate for a given period of time (7-10 days).The colonies formed are then counted and scored for stem cell markerexpression as an indicator of stemness of the original cell. Optionally,the colonies that formed are then picked and passaged to test theirself-renewal and differentiation potential. In a self-renewal assay,when cultured in appropriate culture media, the cells should maintainstem cell marker (e.g., Lgr5) expression over at least one (e.g., 1, 2,3, 4, 5, 10, 20, etc.) cell divisions.

“Subject” includes humans and mammals (e.g., mice, rats, pigs, cats,dogs, and horses). In many embodiments, subjects are mammals,particularly primates, especially humans. In some embodiments, subjectsare livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. In someembodiments (e.g., particularly in research contexts) subject mammalswill be, for example, rodents (e.g., mice, rats, hamsters), rabbits,primates, or swine such as inbred pigs and the like.

“Synergy” or “synergistic effect” is an effect which is greater than thesum of each of the effects taken separately; a greater than additiveeffect.

“TgfBeta inhibitor” (Tgf-β inhibitor) as used herein is a substance thatreduces activity of the TgfBeta pathway. An example of a TgfBetainhibitor can be a TgfBeta receptor inhibitor, which may include but isnot limited to Alk4, Alk7 and Alk5/TgfBeta-RI.

“Tissue” is an ensemble of similar cells from the same origin thattogether carry out a specific function.

“Treating” as used herein in connection with a cell population meansdelivering a substance to the population to effect an outcome. In thecase of in vitro populations, the substance may be directly (or evenindirectly) delivered to the population. In the case of in vivopopulations, the substance may be delivered by administration to thehost subject.

“Valproic acid” (VPA) has chemical formula C₈H₁₆O₂ and the followingalternate name: 2-propylpentanoic acid. The sodium salt of valproic acidcan also be used in place of VPA, and the term “VPA” is usedinterchangeable herein to refer to VPA or pharmaceutically acceptablesalts thereof, such as the sodium salt. Its chemical structure is asfollows:

“Wnt activation” as used herein in connection with a substance orcomposition is an activation of the Wnt signaling pathway.

“Wnt activator” as used herein refers to a substance that activates theWnt signaling pathway.

“Wnt inhibitor” as used herein refers to a substance that inhibits theWnt signaling pathway, which can be for example a Wnt receptorinhibitor, Wnt receptor antagonist, a Porcupine inhibitor which inhibitsWnt secretion, or a Tankyrase inhibitor, or a drug that interfere withβ-catenin interactions. Examples of Wnt inhibitors include Wnt-059,IWP-2, IWR-1-endo, AZ6102, FH535, WIKI4, ICG-001, XAV-939, PRI-724,LGK-974, YA1797K, KY02111, Cardionogen 1, and IWP 12, etc.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein “pharmaceutically acceptable carrier, diluent orexcipient” includes without limitation any adjuvant, carrier, excipient,glidant, sweetening agent, diluent, preservative, dye/colorant, flavorenhancer, surfactant, wetting agent, dispersing agent, suspending agent,stabilizer, isotonic agent, solvent, surfactant, or emulsifier which hasbeen approved by the United States Food and Drug Administration as beingacceptable for use in humans or domestic animals. Examples ofpharmaceutically acceptable carriers include, but are not limited to, tosugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate;tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal andvegetable fats, paraffins, silicones, bentonites, silicic acid, zincoxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesameoil, olive oil, corn oil and soybean oil; glycols, such as propyleneglycol; polyols, such as glycerin, sorbitol, mannitol and polyethyleneglycol; esters, such as ethyl oleate and ethyl laurate; agar; bufferingagents, such as magnesium hydroxide and aluminum hydroxide; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; ethylalcohol; phosphate buffer solutions; and any other compatible substancesemployed in pharmaceutical formulations.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.For example, inorganic salts include, but are not limited to, ammonium,sodium, potassium, calcium, and magnesium salts. Salts derived fromorganic bases include, but are not limited to, salts of primary,secondary, and tertiary amines, substituted amines including naturallyoccurring substituted amines, cyclic amines and basic ion exchangeresins, such as ammonia, isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Example organic bases used in certain embodiments includeisopropylamine, diethylamine, ethanolamine, trimethylamine,dicyclohexylamine, choline and caffeine.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions(e.g., pharmaceutical compositions).

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

DETAILED DESCRIPTION

A description of example embodiments of the disclosure follows.

The present disclosure relates to methods for forming enteroendocrinecells (EECs) from a mammalian post-natal cell population, such as apost-natal stem cell population, by treating with a plurality of smallmolecules that are capable of activating and/or deactivating pathwaysand mechanisms that lead to differentiation of the post-natal cellpopulation into EECs. The pathways and mechanisms acted on by the smallmolecules can include, but are not limited to, Wnt signaling pathways,Notch signaling pathways, EGFR pathways, MEK/ERF signaling pathways,mechanisms acted on by growth factors, Histone Methylation pathways, theTgf-β signaling pathway, and NeuroD1 pathways. In particular, aspects ofthe disclosure provide small molecule combinations that can promotedifferentiation of the post-natal cells such as post-natal stem cells,into EECs, as well as signal the differentiation into a particular EECcell type.

The present disclosure also relates to methods for increasing theinsulin expression in a population of mammalian cells, by treating thepopulation with a plurality of small molecules that are capable ofactivating and/or deactivating pathways and mechanisms that lead toincreased insulin expression on the cells. The cells treated by thesmall molecule can be either post-natal cells (such as post-natal stemcells or multipotent progeny cells) or enteroendocrine cells, such asEECs that have been differentiated from post-natal cells such aspost-natal stem cells, by the method described above. The pathways andmechanisms acted on by the small molecules to increase the insulinproduction can include, but are not limited to, any of the signalingpathways discussed for EEC differentiation above, such as for exampleWnt and Notch signaling pathways, and in particular including smallmolecules that act to inhibit and/or activate DNA methylation pathways,Tgf-β signaling pathways and NeuroD1 pathways. In particular, aspects ofthe disclosure provide for small molecule combinations that promoteinsulin expression in the cells, and that induce cells in the treatedpopulation to become insulin producing cells.

In differentiating the post-natal cells such as post-natal stem cells toEECS, the plurality of small molecules is provided such that expressionof the differentiation marker ChgA is upregulated due to differentiationof the cells. The expression of ChgA is upregulated such that thefraction of cells expressing ChgA in the cell population that has beentreated with the small molecules, as measured in a ChgA ImmunostainingAssay, is at least about 1.5%. The fraction of cells expressing ChgA asmeasured by the ChgA Immunostaining Assay may even be at least about10%, and even at least about 50%, such as a fraction of cells is in arange of from about 60% to about 100%. In one embodiment, the fractionof cells expressing ChgA may be in a range of from about 1.5% to about100%. Furthermore, as measured by an mRNA ChgA Expression Assay, theChgA mRNA expression in the differentiated cell population may be atleast about 10 times the ChgA mRNA expression in the initial post-natalcell population. The ChgA mRNA expression may even be at least 100 timesthe ChgA mRNA expression in the initial post-natal cell population, andmay even be in the range of from about 1000 times to 1,000,000 times theChgA mRNA expression in the initial post-natal cell population. In oneembodiment, the ChgA mRNA expression may be in a range of from about 10to about 1,000,000 times the number of cells expressing ChgA in theinitial post-natal cell population.

The differentiation of the cells to form the EECs using the smallmolecule can provide a highly pure population of enteroendocrine cells,with a significant number of the post-natal cells, such as post-natalstem cells, being converted to the enteroendocrine cells. This abilityto convert the post-natal stem cells into EECs in large quantities issignificant, because intestinal EECs are restricted to the mucosa,predominantly in its deeper half, and typically comprise only a smallminority of the overall epithelial cell population, such as less than1%. In contrast, treatment with the small molecules as described hereinconverts the post-natal cells into the enteroendocrine cells such thatpercentage of cells expressing the ChgA marker for EECs is at least 2%of the total cell population, and even as high as 90% to even 100% ofthe cell population, such as from about 60% to about 90% of the totalcell population, and even from about 70% to about 80% of the total cellpopulation. As disclosed herein, a final cell population having about80% EECs has been achieved (see, e.g., FIG. 40). In one embodiment, thepercentage of cells expressing ChgA in the obtained cell population isincreased as compared to the initial post-natal cell population is anincrease in the fraction of ChgA expressing cells of from at least 0.1%to at least 1%, and even an increase of at least about 5%, such as anincrease of at least about 10%, and even an increase of at least about50%, such as at least about 100%.

The post-natal cells that are treated by the small molecules includepost-natal stem cells that are non-embryonic stem cells, such as adultstem cells that have the capacity to self-renew and to differentiateinto multiple cell lineages, and can also include multipotent progenycells and/or progenitor cells. The post-natal stem cells can includeintestinal stem cells, epithelial stem cells, hematopoietic stem cells,mammary stem cells, mesenchymal stem cells, endothelial stem cells andneural stem cells, which often have conserved signaling and developmentpathways. In one embodiment, the post-natal stem cells are intestinalstem cells, and may be identified by the marker Lgr5. Lgr5 is aLeucine-rich repeat-containing G-protein coupled receptor 5, also knownas G-protein coupled receptor 49 (GPR49) or G-protein coupled receptor67 (GPR67), and in humans is encoded by the Lgr5 gene. When treatingcells with small molecules to increase insulin expression, the cellsbeing treated may be either these post-natal stem cells and/or cellsthat have been further specified, such as multipotent progeny cellsand/or progenitor cells and/or enteroendocrine cells.

Treatment of a population of cells with a plurality of small moleculescan also be performed to provide a significant increase in the insulinexpression of the cells. In one embodiment, the insulin expression canbe increased by treatment with the small molecules such that, asmeasured by an Insulin Activity Assay, the fraction of cells having theInsulin GFP reporter activated in the treated cells is at least about1%, and even at least about 20%. In one embodiment, the fraction ofcells having the Insulin GFP reporter activated is in a range of fromabout 20% to about 100%. According to a different type of measure, thetreatment with the plurality of small molecules may be capable ofincreasing the insulin expression with respect to the initial cellpopulation, as determined by an mRNA Insulin Expression Assay, to alevel substantially greater than a baseline level. For example, incomparing with Hprt mRNA (as a reference gene for expression levels inqPCR), the mRNA Insulin Expression Assay may provide a mRNA insulinexpression level that is in the range of at least about 1 fold and evenat least about 2 fold as compared to a standard using Hprt mRNA levels(where the insulin level of islet cells is typically about 100 fold ofthe Hprt level), thus demonstrating that the cells are beingsuccessfully converted into insulin expressing cells. As shown in FIG.31 herein, a 2×10⁷ fold increase in insulin expression can be achievedas compared to the expression in stem cells, when an arbitrary controlCt value is set for stem cell samples in qPCR (in a case where InsulinmRNA is undetectable at or above the set Ct value in stem cell samples).Furthermore, an insulin mRNA expression level as measured by an mRNAInsulin Expression Assay as compared to insulin mRNA levels in isletcells, is believed to provide at least about 0.01%, such as at leastabout 0.1%, an even at least about 1%, up to at least about 10%, andeven 100% or 200% of the insulin mRNA activity as compared to theislets. According to one embodiment, subjecting a cell population totreatment with the plurality of small molecules can provide a resultingcell population where at least about 1% of the obtained cells areinsulin producing cells, and the number of insulin producing cells inthe obtained cell population may even be in a range of from about 1% toabout 20% of the total cell population.

Treatment of a population of EECs with a plurality of small moleculescan also be performed to generate subset populations comprising, forexample, L-cells, K-cells, I-cells, G-cells, EC-cells, N-cells, andS-cells.

The population of mammalian cells treated by the small molecules can bean in vitro population, such as a population of cells dispersed in acell culture medium. An in vitro population of cells may also be in theform of organoids, which is a cell cluster or aggregate that resemblesan organ, or part of an organ, and possesses cell types relevant to thatparticular organ (Sato, 2009). Alternatively, the population ofmammalian cells treated by the small molecules may be an in vivopopulation, such as in vivo treatment of a human or other mammal totreat a disease state. In another alternative, the population ofmammalian cells treated by the small molecules may be an ex vivopopulation, such as a population of cells obtained (e.g., isolated,derived) from a human or other mammal (e.g., a human or other mammal inneed of treatment). A proposed mechanism for one embodiment for thedifferentiation of EECs from post-natal cells such as post-natal stemcells, is provided in FIG. 34. Without being limited by any particulartheory, it is presently believed that by controlling the Wnt and Notchpathways, and optionally other pathways, the EECs can be specified fromthe stem cells. In particular, in the mechanism as shown provides foractivating the Wnt pathway while inhibiting the Notch pathway, and theninhibiting both the Notch and Wnt pathways to form EECs. Othermechanisms and/or pathways may also be activated and/or inhibited, asdescribed below, to further specify differentiation along the pathwaytowards EECs (as opposed to goblet cells, enterocytes, Paneth cells,etc.), and other mechanisms and/or pathways may also be used to furtherspecify particular types of EECs, and/or EEC function. For example,while FIG. 34 shows Wnt inhibition, in some embodiments it may bepossible to provide the differentiation to EEC without requiringaddition of a Wnt inhibitor. Also, while FIG. 34 illustrates a two stagemethod for obtaining EECS, certain embodiments may also provide for justa single stage of treatment, or even 3 or more stages of treatment toachieve the EECs. Particular types of EECs that might be specified caninclude cells that express GLP-1, SHT, SST, gastrin, CCK, SCT, NTS, PYY,and ghrelin, among others.

In embodiments of the method of increasing insulin production ofmammalian cells as described herein, treatment of the mammalian cellsmay result in differentiation along a similar pathway as that shown inFIG. 34, such as from post-natal stem cells to EECs, with the resultingcells being those that provide increased insulin production, and/or thetreatment may be performed on EECs themselves, to convert these cellsinto cells with increased insulin production. That is, the treatment toincrease insulin production of the cells can be performed simultaneouslywith, before, or after differentiation of post-natal stem cells intoEECS, or can be performed on cells at a differentiation stage in betweenstem cells and EECS, as well on the post-natal stem cells or EECsthemselves. Accordingly, the treatment to increase insulin productioncan be performed on a mammalian cell population that comprises at leastone of post-natal stem cells (such as Lgr5+ cells), progenitor cells anddifferentiated enteroendocrine cells.

FIG. 34 also shows the different types of intestinal cells, such asEECs, enterocytes, goblet cells, Paneth cells and stem cells and ageneral configuration of such cells as found in the crypts of theintestine. This figure shows that, as discussed above, theenteroendocrine (EEC) cells are typically located in such crypts, andare only present in a small percentage of the total cells therein (<1%),and thus it has been difficult to test mechanisms for disease treatmentin the absence of an in vitro method for obtaining an EEC population ofcells.

As discussed above, in one embodiment the plurality of small moleculesprovided to treat the post-natal cell population (such as a post-natalstem cell population) to achieve differentiation to EECs, and/or totreat a cell population to increase the insulin expression thereof,includes a small molecule that acts to activate and/or inhibit the Wntpathway. The Wnt signaling pathway is a signal transduction pathwayactivated by binding of a Wnt protein ligand to a Frizzled familyreceptor. Wnt activators that can be provided as a part of a treatmentcan include, for example, at least one of R-Spondin 1, Wnt3a, Gskinhibitors such as at least one of CHIR99021, LY2090314, NP031112(Tideglusib), lithium, A1070722, SKL2001, and other agents capable ofactivating the Wnt signaling pathway. In one embodiment, a Wnt activatorprovided as a part of a plurality of small molecules can be R-Spondin 1,which is a protein belonging to the R-Spondin family. Wnt inhibitorsthat can be provided as a part of a treatment can include, for example,at least one of Wnt-059, Dkk family proteins (such as at least one ofDkk-1, Dkk2, Dkk3 and Dkk4), sFRPs (such as at least one of sFRP-1 andsFRP-2), antibodies against Wnt receptors such as OMP-18R5, other smallmolecule Wnt inhibitors such as LGK974, CWP232291, PRI-724, IQR-1, IWP2,IWP-L6, ICG-001, WIKI4, Ky02111, FH535, XAV939, NSC668036, FJ9,3289-8625, and others (Kahn, 2014). In one embodiment, a Wnt inhibitorprovided as a part of a plurality of small molecules for treatment canbe Wnt-059, which has the following chemical structure:

Derivatives and/or pharmaceutically acceptable salts of the Wntactivator and/or Wnt inhibitor may also be provided.

Further Examples of Wnt agonists which may be suitable as Wnt activatorscan be found in the following Table.

TABLE Wnt Agonist Column A Column B Wnt Ligand Wnt1/Int-1 Wnt-2/Irp(Int-I-related protein) Wnt-2b/13 Wnt-3/Int-4 Wnt-3a Wnt-4 Wnt-5a Wnt-5bWnt-6 Wnt-7a Wnt-7b Wnt-8a/8d Wnt-8b Wnt-9a/14 Wnt-9b/14b/15 Wnt-10aWnt-10b/12 Wnt-11 Wnt-16 Wnt Related Protein R-Spondin 1/2/3/4 NorrinGSK3b inhibitor Other Wnt modulator (hetero)arylpyrimidines Wnt AgonistIQ 1 DCA QS 11 WASP-1, ZINC00087877 WAY 316606, HCl WAY-262611, HClHLY78 SKL2001 Cpd1 Cpd2 cmpd 109 ISX 9 Cmpd 71 Cmpd 2 Selumetinib(AZD6244) Radicicol (hetero)arylpyrimidines Wnt Agonist WAY 316606, HClWAY-262611, HCl SKL2001 ISX 9

In one embodiment the plurality of small molecules provided to treat thepost-natal cell population to achieve differentiation to EECs, and/or totreat a cell population to increase the insulin expression thereof,includes a small molecule that acts to inhibit the Notch pathway. Notchinhibitors that can be provided as a part of a treatment can include,for example, at least one of DAPT; LY411575; MDL-28170; R04929097;L-685458((5S)-(t-Butoxycarbonylamino)-6-phenyl-(4R)hydroxy-(2R)benzylhexanoyl)-L-leu-L-phe-amide);BMS-708163 (Avagacestat); BMS-299897(2-[(1R)-1-[[(4-Chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoicacid); M-0752; YO-01027; MDL28170 (Sigma); LY41 1575(N-2((2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl)-N1-((7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)-1-alaninamide);ELN-46719 (2-hydroxy-valeric acid amide analog of LY41 1575; PF-03084014((S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide);Compound E((2S)-2-{[(3,5-Diflurophenyl)acetyl]amino}-N-[(3S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-1,4-benzodiazepin-3-yl]propanamide;and Semagacestat (LY450139; (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-{[(1S)-3-methyl-2-oxo-2,3,4,5-tetrahydro-1H-3-benzazepin-1-yl]amino}-2-oxoethyl)butanamide).In one embodiment, a Notch inhibitor provided as a part of a pluralityof small molecules can be DAPT, also known asN—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.Derivatives and/or pharmaceutically acceptable salts of the Notchinhibitor may also be provided.

The plurality of small molecules provided for treatment may also includea small molecule that acts to inhibit the MEK/ERK pathway. This caninclude inhibitors of the core Ras-Raf-MEK-ERK signaling cascade, aswell as inhibitors of proteins upstream or downstream of this coresignaling cascade, such as EGF receptor (EGFR) inhibitors. MEK/ERKinhibitors provided for treatment can include, for example, at least oneof PD0325901, AZD8330 (ARRY-424704), Refametinib (BAY 86-9766, RDEA119),Cobimetinib (GDC-0973, XL-518, RG7421), E6201, MEK162 (ARRY-438162),Pimasertib (AS703026, MSC1936369B), R04987655 (CH4987655), RO5126766(CH5126766), Selumetinib (AZD6244, ARRY-142,886), TAK-733, Trametinib(GSK1120212), and GDC-0623, WX-554 (Zhao and Adjei, 2014), and may alsoand/or alternatively include EGFR inhibitors such as Erlotinib,Gefitinib, Lapatinib, Afatinib, Neratinib, AZ5104, Afatinib, PD 153035,CL-387785, AST-1306, PD 168393, Canertinib, and other EGFR inhibitors,as well as Ras and Raf inhibitors. In one embodiment, a MEK/ERKinhibitor provided as a part of a plurality of small molecules can bePD0325901, also known asN-[(2R)-2,3-Dihydroxypropoxyl]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide.Derivatives and/or pharmaceutically acceptable salts of the MEK/ERKinhibitor may also be provided.

The plurality of small molecules provided for treatment may also includea small molecules corresponding to various growth factors, such as atleast one of epidermal growth factor (EGF) and Noggin. In oneembodiment, the growth factors provided as a part of the small moleculesfor treatment include EGF and/or Noggin. Derivatives and/orpharmaceutically acceptable salts of the growth factors may also beprovided. In another embodiment, the plurality of small molecules caninclude an HDAC Inhibitor (Histone deacetylase inhibitor). An HDACinhibitor that can be provided as a part of the plurality of smallmolecules for treatment can include Tubastatin A, ACY1215, Valproicacid, SAHA, Trichostatin A, SHBA, CBHA, LAQ-824, PDX-101, LBH-589,ITF2357, PCI-24781, Compound 7 (ChemieTek), JNK-24681585 (Quisinostat)SB939 (Pracinostat), 4SC-201 (Resminostat), Tefinostat (CHR-2845),CHR-3996, CG200745, Depsipeptide (Romidepsin), Butyrate, MS-275,MGCD0103 and CI994, among others. In one embodiment, an HDAC inhibitorprovided for treatment can be Tubastatin A, also calledN-Hydroxy-4-(2-methyl-1,2,3,4-tetrahydro-pyrido[4,3-b]indol-5-ylmethyl)benzamidehydrochloride. Derivatives and/or pharmaceutically acceptable salts ofthe HDAC inhibitor may also be provided.

Further examples of HDAC inhibitors can be found in the following Table.

TABLE HDAC Inhibitors Column A Column B Class Agent HydroxamatesTrichostatin A (TSA) Hydroxamates SAHA (Zolinza, vorinostat)Hydroxamates 4-iodo-SAHA Hydroxamates SBHA Hydroxamates CBHAHydroxamates LAQ-824 Hydroxamates PDX-101 (belinostat) HydroxamatesLBH-589 (panobinostat) Hydroxamates ITF2357 (Givinostat) HydroxamatesPCI-34051 Hydroxamates PCI-24781 (Abexinostat) Hydroxamates Tubastatin AHydroxamates CUDC-101 Hydroxamates Compound 7 Hydroxamates OxamflatinHydroxamates ITF2357 Hydroxamates Bufexamac Hydroxamates APHA Compound 8Hydroxamates JNJ-26481585 (Quisinostat) Hydroxamates Suberoylanilide-d5Hydroxamic Acid Hydroxamates HDAC Inhibitor XXIV Hydroxamates TubacinHydroxamates Butyrylhydroxamic acid Hydroxamates 1-NaphthohydroxamicAcid Hydroxamates MC 1568 Hydroxamates SB939 (Pracinostat) Hydroxamates4SC-201 (Resminostat) Hydroxamates Tefinostat (CHR-2845) HydroxamatesCHR-3996 Hydroxamates CG200745 Cyclic peptide Depsipeptide (Romidepsin,FK-228, FR 901228) Cyclic peptide Trapoxin A Cyclic peptide HC ToxinAliphatic Acid Valproic Acid Aliphatic Acid VAHA Aliphatic Acid Phenylbutyrate Aliphatic Acid Butyrate Aliphatic Acid AN-9 Benzamides MS-275(Entinostat) Benzamides MGCD0103 (Mocetinostat) Benzamides CI994(Tacedinaline; PD-123654; GOE-5549; Acetyldinaline) Benzamides BML-210Hydroxamates M 344 Benzamides Chidamide Hydroxamates4-(dimethylamino)-N-[6-(hydroxyamino)-6- oxohexyl]-benzamideMiscellaneous Luteolin Prodrug of thiol PTACH Miscellaneous L-CarnitineMiscellaneous Biphenyl-4-sulfonyl chloride Miscellaneous SIRT1/2Inhibitor VII Hydroxamates (S)-HDAC-42 Hydroxamates Indole-3-acetamideMiscellaneous NSC 3852 Miscellaneous PPM-18 Miscellaneous Ratjadone A,Synthetic Benzamides N-(2-Aminophenyl)-N′-phenylheptanediamideMiscellaneous Dihydrochlamydocin Miscellaneous 7-AminoindoleMiscellaneous Apicidin Miscellaneous Parthenolide Hydroxamates HNHAMiscellaneous Splitomicin Benzamides RGFP109 Benzamides RGFP136Benzamides RGFP966 Benzamides 4SC-202 Hydroxamates ACY1215 MiscellaneousME-344 Miscellaneous Sulforaphane CF3Methyl Ketones 6H CF3Methyl Ketones27 Aryl Ketones 25 Non classical 5 Nexturastat A Droxinostat AR-42Romidepsin (FK228, Depsipeptide) Scriptaid Sodium Phenylbutyrate TMP269Thailandepsin A BRD9757 LMK235 HPOB CAY10603 Tasquinimod HDAC6 InhibitorIII HDAC Inhibitor XXIV HDAC Inhibitor IV HDAC Inhibitor XIX HDACInhibitor XXII HDAC Inhibitor VII HDAC Inhibitor II HDAC Inhibitor VI(−)-Depudecin KD 5170 TC-H 106 TCS HDAC6 20b Pyroxamide ChidamideHDAC-IN-1 HC Toxin Hydroxamates SAHA (Zolinza, vorinostat) HydroxamatesLBH-589 (panobinostat) Hydroxamates JNJ-26481585 (Quisinostat) Cyclicpeptide Depsipeptide (Romidepsin, FK-228, FR 901228) Benzamides MGCD0103(mocetinostat) Prodrug of thiol PTACH Miscellaneous Ratjadone A,Synthetic Miscellaneous Apicidin CF3Methyl Ketones 27 Non classical 5Nexturastat A Droxinostat Scriptaid BRD9757 HPOB CAY10603 HDAC6Inhibitor III Hydroxamates ACY1215 Hydroxamates Tubastatin AHydroxamates Tubacin Hydroxamates Trichostatin A (TSA)

A Histone Methylation inhibitor (e.g., Histone demethylase (HDM)inhibitor) that can be provided as a part of the plurality of smallmolecules for treatment can include at least one of Tranylcypromine,GSK-2879552, GSK-LSD1, SP-2509, GSK J4, 2,4-Pyridinedicarboxylic Acid,ML324, IOX 1, OG-L002, CBB1007, GSK J1, GSK J2, and GSK J5, amongothers. In one embodiment, a Histone Methylation inhibitor provided fortreatment can be Tranylcypromine. In an embodiment, a HistoneMethylation inhibitor provided for treatment can be a lysine-specifichistone demethylase (LSD1). Derivatives and/or pharmaceuticallyacceptable salts of the Histone Methylation inhibitor may also beprovided. Examples of Histone Methylation inhibitors include:JmjC-domain demethylase: Jmjd2, Jmjd2C, Jmjd3; Lysine-specificdemethylase: LSD1 inhibitors such as Tranylcypromine (LSD1), RN 1 (LSD1), GSK2879552 (LSD1), CBB1003 (LSD1), OG-L002 (LSD1), CBB1007 (LSD1),2,4-Pyridinedicarboxylic Acid (LSD), SP2509 (LSD1), ORY-1001 (RG-6016),GSK LSD1 (LSD1); Jmjd: Daminozide (Jmjd), GSK J1 (Jmjd3/UTX), GSK J4(Jmjd3), IOX 1 (Jmjd), JIB 04 (Jmjd), NSC 636819 (KDM4A/KDM4B), TC-E5002 (KDM2/7), Pargyline, ML324 (Jmjd2);

A Tgf-β inhibitor that can be provided as a part of the plurality ofsmall molecules for treatment can include at least one of 616452(Repsox), LY-364947, SB-505124, A-83-01, SB-431542, TGF-βRI KinaseInhibitor VII, SB-525334, TGF-βRI Kinase Inhibitor IX, GW788388,LY2109761, Galunisertib (LY2157299), EW-7197, Pirfenidone, K02288, D4476, R 268712, A77-01, and SM16, as well as antibodies against Tgf-βreceptors. In one embodiment, a Tgf-β inhibitor provided for treatmentcan be 616452, also referred to as RepSox, with the chemical formula2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5naphthyridine.Derivatives and/or pharmaceutically acceptable salts of the HistoneMethylation inhibitor may also be provided.

Further examples of Tgf-β inhibitors can be found in the following Table

TGF-β Inhibitors Class Agent Alternative Name Tgf-beta-R1 inhibitorLY-364947 616451, TGF-β RI Kinase Inhibitor I, CAS 396129- 53-6,[3-(Pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, ALK5 Inhibitor I,LY-364947, HTS-466284 Tgf-beta-R1 inhibitor Repsox 616452, TGF-β RIKinase Inhibitor II, CAS 446859- 33-2,2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)- 1,5-naphthyridineTgf-beta-R1 inhibitor SB-505124 616453, TGF-β RI Kinase Inhibitor III,CAS 356559- 13-22-(5-Benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl, ALK5 Inhibitor III, SB-505124, HClTgf-beta-R1 inhibitor A-83-01 616454, TGF-β RI Kinase Inhibitor IV - CAS909910- 43-6, 3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole, A-83-01, ALK5 Inhibitor IV Tgf-beta-R1inhibitor SD-208 616456, TGF-β RI Kinase Inhibitor V, CAS 627536- 09-8,2-(5-Chloro-2-fluorophenyl)pteridin-4- yl)pyridin-4-yl amine, SD-208,ALK5 Inhibitor V Tgf-beta-R1 inhibitor SB-431542 616461, TGF-β RI KinaseInhibitor VI, SB431542 - CAS 301836-41-9, 4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol- 2-yl]benzamide,Dihydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide, Dihydrate Tgf-beta-R1inhibitor TGF-β RI Kinase 616458, TGF-β RI Kinase Inhibitor VII - CASInhibitor VII 666729-57-3, 1-(2-((6,7-Dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone, ALK5 Inhibitor VIITgf-beta-R1 inhibitor SB-525334 616459, TGF-β RI Kinase Inhibitor VIII -CAS 356559-20-1, SB-525334, 6-(2-tert-Butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)- quinoxaline, ALK5 Inhibitor VIIITgf-beta-R1 inhibitor TGF-β RI Kinase 616463, TGF-β RI Kinase InhibitorIX, 4-((4-((2,6- Inhibitor IX Dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide, ALK5 Inhibitor IX Tgf-beta-R1 inhibitorGW788388 4-(4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridin-2-yl)-N-(tetrahydro-2H-pyran-4-yl)benzamide Tgf-beta-R1 inhibitor LY21097617-(2-morpholinoethoxy)-4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline Tgf-beta-R1 inhibitorGalunisertib 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H- (LY2157299)pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6- carboxamide Tgf-beta-R1inhibitor EW-7197 N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2- methanamine Tgfbproduction Pirfenidone 2(1H)-Pyridinone, 5-methyl-1-phenyl- inhibitorTgf-beta-R1 inhibitor K02288 3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol Tgf-beta-R1 inhibitor D 44764-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide Tgf-beta-R1 inhibitor R 2687124-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol Other ITD 14-[1,1′-Biphenyl]-4-yl-1,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3-quinolinecarboxylic acid ethyl ester Smad3 inhibitorSIS3 1,2,3,4-Tetrahydro-6,7-dimethoxy-2-[(2E)-3-(1-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-1-oxo-2- propenyl]-isoquinolinehydrochloride Tgf-beta-R1 inhibitor A77-014-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4- yl]quinoline OtherAsiaticoside Tgf-beta-R1 inhibitor SM164-(5-(benzo[d][1,3]dioxol-5-yl)-4-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)bicyclo[2.2.2]octane-1- carboxamide Tgf-betaantibody ID11 Tgf-beta antibody 2G7 Tgf-beta antibody GC-1008Fresolimumab Tgf-beta antibody CAT-152 Lerdelimimab Tgf-beta antibodyCAT-192 Metelimumab TGf-beta Receptor PF-03446962 antibody Tgf-betaantibody SR-2F Tgf-beta antibody 2G7 Tgf-beta antibody LY2382770Tgf-beta antibody IMC-TR1 Tgf-beta antibody STX-100 TGF-beta antagonistTGF-PRII: Fc Recombinant protein betaglycan/TGF- PRIII OligonucleotideAP12009 Trabedersen, antisense molecule inhibitor OligonucleotideAP11014 inhibitor Oligonucleotide AP15012 inhibitor LY-550410 LY-580276LY-364947 LY-2109761 LY-2157299 Galunisertib LY-573636 Is this TGF binhibitor/YES SB- 505124 SB-431542 SB-525234 SD-208 SD-093 Ki-26894NPC-30345 SX-007 IN-1130 pyrrole-imidazole Gene siliencing polyamideEW-7203 EW-7195 Structure EW-7197 GW6604 U.S. Pat. No. Pyrrolederivatives as pharmaceutical agents 7,087,626 U.S. Pat. No. Quinazolinederivatives as medicaments 6,476,031 U.S. Pat. No. Antibodies to TGF-β7,723,486, and EP 0945464 Peptide Trx-xFoxHIb Smad-interacting peptideaptamers Peptide Trx-Lefl Peptide Distertide (pI44) Peptide pI7 PeptideLSKL dihydropyrrlipyrazole- See US Patent U.S. based scaffold Pat. No.8,298,825 B1 imidazole-based See US Patent U.S. scaffold Pat. No.8,298,825 B1 pyrazolopyridine-based See US Patent U.S. scaffold Pat. No.8,298,825 B1 pyrazole-based scaffold See US Patent U.S. Pat. No.8,298,825 B1 imidazopyridine-based See US Patent U.S. scaffold Pat. No.8,298,825 B1 triazole-based scaffold See US Patent U.S. Pat. No.8,298,825 B1 pyridopyrimidine-based See US Patent U.S. scaffold Pat. No.8,298,825 B1 pyrrolopyrazole-based See US Patent U.S. scaffold Pat. No.8,298,825 B1 isothiazole-based See US Patent U.S. scaffold Pat. No.8,298,825 B1 oxazole-based scaffold See US Patent U.S. Pat. No.8,298,825 B1 Tgf-beta-R1 inhibitor Repsox 616452, TGF-β RI KinaseInhibitor II, CAS 446859- 33-2,2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)- 1,5-naphthyridineTgf-beta-R1 inhibitor Galunisertib4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H- (LY2157299)pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6- carboxamide Tgf-beta-R1inhibitor EW-7197 N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2- methanamine Tgfbproduction Pirfenidone 2(1H)-Pyridinone, 5-methyl-1-phenyl- inhibitorLY-2157299 Galunisertib Tgf-beta-R1 inhibitor SB-505124 616453, TGF-β RIKinase Inhibitor III, CAS 356559-13-22-(5-Benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl, ALK5 Inhibitor III, SB-505124, HClTgf-beta-R1 inhibitor SB-525334 616459, TGF-β RI Kinase Inhibitor VIII -CAS 356559-20-1, SB-525334, 6-(2-tert-Butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)- quinoxaline, ALK5 Inhibitor VIIITgf-beta-R1 inhibitor TGF-β RI Kinase 616463, TGF-β RI Kinase InhibitorIX, 4-((4-((2,6- Inhibitor IX Dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide, ALK5 Inhibitor IX Tgf-beta-R1 inhibitor R268712 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol SB- 505124 Pyridine,2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-, hydrochloride (1:1) SD-208IN-1130 EW-7197 Tgf-beta-R1 inhibitor A-83-01 616454, TGF-β RI KinaseInhibitor IV - CAS 909910- 43-6,3-(6-Methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole, A-83-01, ALK5 Inhibitor IV Tgf-beta-R1inhibitor SB-431542 616461, TGF-β RI Kinase Inhibitor VI, SB431542 - CAS301836-41-9, 4-[4-(3,4- Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide, Dihydrate, 4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]benzamide, Dihydrate Tgf-beta-R1inhibitor R 268712 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol

A NeuroD1 activator that can be provided as a part of the plurality ofsmall molecules for treatment can include ISX-9 and other Isoxazolemolecules (Schneider, 2008). In one embodiment, the NeuroD1 activatorprovided for treatment can be ISX-9, with the chemical formulaN-cyclopropyl-5-(2-thienyl)-3-isoxazolecarboxamide. Derivatives and/orpharmaceutically acceptable salts of the Neuro D1 activator may also beprovided.

The plurality of small molecules provided for treatment may also includeone or more monoamine oxidase (MAO) inhibitors. Examples of MAOinhibitors include Safinamide Mesylate, Rasagiline Mesylate,Tranylcypromine, Moclobemide (Ro 111163), Isatin,8-(3-Chlorostyryl)caffeine, Bifemelane hydrochloride, (R)-(−)-Deprenylhydrochloride, Harmane, Lazabemide hydrochloride, Pirlindole mesylate,RN 1 dihydrochloride, and Tetrindole mesylate, among others.

The plurality of small molecules provided for treatment may also includeone or more BMP receptor (including ALK2) inhibitors. Example of BMPreceptor (including ALK2) inhibitors include DMH-1, DMH2, Dorspmrphin, K02288, LDN 193189, and ML 347.

In the treatment of cells to increase insulin expression, a plurality ofsmall molecules that has been shown to be effective in increasinginsulin expression of cells is a plurality of small molecules comprisingat least one of a DNA methylation inhibitor, a Tgf-β inhibitor, and aNeuroD1 activator. In one embodiment, all three small molecules areprovided together as a part of a treatment to increase insulinproduction in a cell population. The Tgf-β inhibitor and NeuroD1activator may be any of those discussed above, and/or their derivativesand/or the pharmaceutically acceptable salts thereof, and in particularmay correspond to 616452 and ISX-9. The structure of 616452 (Repsox) isas follows:

The DNA Methylation inhibitor can comprise at least one of5-Azacytidine, 5-Aza-2-deoxycytidine, RG 108, SGI 1027, Nanaomycin A,Zebularine, Lomeguatrib, SGI-110, and Nanaomycin C, among others.Examples of DNA methylation inhibitors are described in U.S. Pat. No.8,207,142, Canada Patent No. 2,454,147, and WO 2012/087889, each ofwhich is specifically incorporated herein by reference. In oneembodiment, the DNA Methylation inhibitor comprises 5-Azacytidine.Derivatives and/or pharmaceutically acceptable salts of the DNAMethylation inhibitor may also be provided. Additional examples of DNAmethylation inhibitors (e.g., DNA methyl transferase (DNMT) inhibitors)include: DNA analog 5-Azacytidine, Zebularine, Decitabine; DNMTinhibitor: Caffeic acid purum, Chlorogenic acid, (−)-Epigallocatechingallate (EGCG), Hydralazine, Procainamide, Procaine, Psammaplin A,RG108, Fisetin, Lomeguatrib, SGI 1027, 5-Iodotubercidin, 6-Thioguanine,MG98, DC-05, and DC-517.

Table 1 below presents a list of molecules, their abbreviation asotherwise used in the description herein, and examples of the finalconcentration of each reagent when used in in vitro experimentsdescribed herein.

TABLE 1 Summary of growth factors and small molecules used in studyReagent Name Abbreviation Final Concentration EGF E 50 ng/ml Noggin N100 ng/ml R-Spondin1 R 500 ng/ml CHIR99021 Chir, C 5 μM Valproic AcidSodium Salt VPA, V 1.25 mM DAPT D 5 μM Tubastatin A Tu 10 μMTranylcypromine Tranyl, Ty 2 μM ISX-9 ISX, I 10 μM Wnt-C59 C59, C 5 μMPD0325901 Pd 1 μM 616452/Repsox 6 5 μM 5-Azacytidine 5-Aza, 5 1 μM

According to one embodiment, the treatment performed using the pluralityof small molecules can be performed in several stages, such as a firstand second stage, or even more stages. The number of stages used fortreatment can be selected according to the differentiation and/orinsulin expression increasing method to be performed, as well as withrespect to the type of small molecules being used and the pathways beingactivated and/or inhibited. For example, in a method of treatment todifferentiate post-natal cells such as post-natal stem cells into EECs,a first stage may be performed to contact the post-natal cellspopulation with one or more first small molecules that upregulate Ngn3,and in a second stage the post-natal cell population may be contactedwith one or more second small molecules that downregulate Ngn3 todecrease Ngn3 expression in the cells. The first and second stagesaccording to this embodiment may promote differentiation of thepost-natal cells such as post-natal stem cells towards the formation ofEECs. In an embodiment of a method of increasing insulin, first throughthird stages may be performed to treat cells to upregulate insulin andincrease expression.

According to one aspect, a treatment to differentiate post-natal cellssuch as post-natal stem cells to EECs can comprise first and secondstages, with a first stage including contacting the cells with firstsmall molecules including a Notch inhibitor and a Wnt activator, and thesecond stage including contacting the cells with second small moleculesincluding a Notch inhibitor and at least one of an EGFR inhibitor and aMEK/ERK inhibitor. For example, the first stage may provide R-Spondin1and DAPT, whereas the second stage may provide DAPT and PD0325901. Thesecond stage may also optionally include a Wnt inhibitor, such asWnt-059. The first and second stages may also include one or more growthfactors, such as EGF and/or Noggin.

According to yet another aspect, the first stage further comprisescontacting the cells with at least one of an HDAC inhibitor, a HistoneMethylation Inhibitor, and a NeuroD1 Activator, such as at least one ofTubastatin A, Tranylcypromine and ISX-9. The second stage may furthercomprise contacting the cells with a Tgf-β inhibitor such as 616452,and/or contacting the cells with a histone methylation inhibitor, suchas Tranylcypromine.

In one embodiment, a treatment to increase insulin expression in a cellpopulation can comprise first, second and third stages, with a firststage including contacting the cells with first small moleculesincluding a Notch inhibitor, a Wnt activator and a DNA methylationinhibitor, a second stage including contacting the cells with secondsmall molecules including a Notch inhibitor, optionally a Wnt inhibitor,a Tgf-β inhibitor, and a NeuroD1 activator, and a third stage includingcontacting the cells with third small molecules including a Notchinhibitor, optionally a Wnt inhibitor, a MEK/ERK inhibitor, and a Tgf-βinhibitor. For example, the first stage may provide R-Spondin1, DAPT,and at least one of 5-Azacytidine and 5-Aza-2-deoxycytidine, whereas thesecond stage may provide DAPT, Wnt-059, 616452 and ISX-9, and the thirdstage may provide DAPT, Wnt-059, 6116452, and PD0325901. The first,second and/or third stages may also include one or more growth factors,such as EGF and/or Noggin.

According to yet another aspect, the first stage further comprisescontacting the cells with at least one of an HDAC inhibitor, a HistoneMethylation Inhibitor, and a NeuroD1 Activator, such as at least one ofTubastatin A, Tranylcypromine and ISX-9. The second stage may furthercomprise contacting the cells with a histone methylation inhibitor, suchas Tranylcypromine.

As described herein, the small molecules may be provided in differentcombinations, and in different stages of treatment, to provide fordifferentiation of post-natal cells such as post-natal stem cells toEECs and/or increased expression of insulin. Some embodiments of smallmolecule combinations for treatment are described below.

According to one aspect, a method of differentiation can comprisetreating mammalian post-natal cells such as post-natal stem cells with aplurality of small molecules including a Notch inhibitor (e.g. DAPT), aMEK/ERK inhibitor (e.g., PD0325901) and one or more growth factors(e.g., EGF and Noggin). The treatment can be conducted in a singlephase.

According to another aspect, a method of differentiation can comprise afirst stage with a first set of small molecules including a Notchinhibitor (e.g. DAPT), a Wnt activator (e.g. R-Spondin1), and one ormore growth factors (e.g., EGF and Noggin), and a second stage with aNotch inhibitor (e.g. DAPT), a MEK/ERK inhibitor (e.g., PD0325901), andone or more growth factors (e.g., EGF and Noggin).

According to another aspect, a method of differentiation can comprise afirst stage with a first set of small molecules including a Notchinhibitor (e.g. DAPT), a Wnt activator (e.g. R-Spondin1), and one ormore growth factors (e.g., EGF and Noggin), a HDAC inhibitor (e.g.Tubastatin A) and a Histone Methylation inhibitor (e.g.Tranylcypromine), and a second stage with a Notch inhibitor (e.g. DAPT),a MEK/ERK inhibitor (e.g., PD0325901), and a Histone Methylationinhibitor (e.g. Tranylcypromine).

According to another aspect, a method of differentiation can comprise afirst stage with a first set of small molecules including a Notchinhibitor (e.g. DAPT), a Wnt activator (e.g. R-Spondin1), and one ormore growth factors (e.g., EGF and Noggin), a HDAC inhibitor (e.g.Tubastatin A) and a Histone Methylation inhibitor (e.g.Tranylcypromine), and a second stage with a Notch inhibitor (e.g. DAPT),a MEK/ERK inhibitor (e.g., PD0325901), a Histone Methylation inhibitor(e.g. Tranylcypromine), and a Wnt inhibitor (e.g., WNT-059).

According to another aspect, a method of differentiation can comprise afirst stage with a first set of small molecules including a Notchinhibitor (e.g. DAPT), a Wnt activator (e.g. R-Spondin1), and one ormore growth factors (e.g., EGF and Noggin), a HDAC inhibitor (e.g.Tubastatin A), a Histone Methylation inhibitor (e.g. Tranylcypromine),and a NeuroD1 activator (e.g. ISX9), and a second stage with a Notchinhibitor (e.g. DAPT), a MEK/ERK inhibitor (e.g., PD0325901), a HistoneMethylation inhibitor (e.g. Tranylcypromine), a Wnt inhibitor (e.g.,WNT-059), and a Tgf-β inhibitor (e.g., 616452).

In one embodiment, a method for increasing insulin in a cell populationcan comprise contacting the cells with small molecules including a DNAmethylation inhibitor (e.g. 5-Azacytidine and/or 5-Aza2-deoxycytidine),a Tgf-β inhibitor (e.g. 616452), and a NeuroD1 activator (e.g. ISX-9).The cell population can be contacted with the molecules in a singlestage, or can be contacted with one or more of the molecules in aplurality of stages.

In yet another aspect, a method for increasing insulin in a cellpopulation includes a three stage process including, in a first stage,contacting the cells with a Notch inhibitor (e.g. DAPT), a Wnt activator(e.g. R-Spondin1), and a DNA methylation inhibitor (e.g. 5-Azacytidineand/or 5-Aza2-deoxycytidine), in a second stage contacting the cellswith a Notch inhibitor (e.g. DAPT), a Wnt inhibitor (e.g. Wnt-059), aTgf-β inhibitor (e.g. 616452), and a NeuroD1 activator (e.g. ISX-9), andin a third stage contacting the cells with a Notch inhibitor (e.g.DAPT), a Wnt inhibitor (e.g. Wnt-059), a Tgf-β inhibitor (e.g. 616452),and a MEK/ERK inhibitor (e.g., PD0325901). Growth factors such as EGFand Noggin can also be provided in the first stage, and the HistoneMethylation inhibitor (e.g., Tranylcypromine) can be provided in thefirst and second stages. A HDAC inhibitor (e.g. Tubastatin A) and/or aNeuroD1 activator (e.g. ISX-9) can also be provided in the first stage.

By performing treatment of post-natal cells such as post-natal stemcells to differentiate cells into enteroendocrine cells, a cellpopulation having a relatively high content of EECs can be obtained.According to one aspect, a cell population having enteroendocrine cellsderived from post-natal cells such as post-natal stem cells can beprovided by treatment with the plurality of small molecules, wherein afraction the enteroendocrine cells is at least about 1% of the totalcell population. In another aspect, the fraction of the enteroendocrinecells in the cell population may be at least about 10%, such as from 20%to 100% of the total cell population. The cells of the cell populationmay also be cryopreserved for use in further in vitro or in vivoapplications. The cell population is non-sorted, meaning that the cellpopulation has not been filtered or otherwise sorted to achieve thedensity of the cells. According to one aspect, the differentiated cellsmay be capable of expressing 5-HT and/or GLP-1, as shown in the Examplesincluded herewith.

Also, the method of increasing insulin production described herein canresult in a cell population having a relatively high fraction of insulinproducing cells, such as a fraction of insulin producing cells that isat least about 0.05% of the total cell population, and even at leastabout 1% of the total cell population.

The method for treating cells described herein can be performed by useof kits that provide a cell culture medium, such as matrigel, along witha cell population targeted for the treatment method, and a plurality ofsmall molecules. The kits may also include instructions for using thekit, and other items of equipment to facilitate carrying out thetreatment methods. In one embodiment, a kit includes a cell culturemedium, mammalian post-natal cells such as post-natal stem cells, and aplurality of small molecules that upregulate ChgA and differentiate thepost-natal cells. In another embodiment, a kit includes a cell culturemedium, mammalian cells, and a plurality of small molecules thatincrease insulin production in the cells.

Aspects of the disclosure are further directed to the treatment ofdisease states in a mammal, such as a human, using the treatment methodsdescribed herein. According to one aspect, a disease state characterizedby insufficient endocrine or enteroendocrine cell products is treated,such as at least one of obesity, diabetes, irritable bowel syndrome,infectious enteritis, and inflammatory bowel disease. In another aspect,a disease state characterized by insufficient insulin production istreated, such as at least one of obesity, and Type I diabetes. Treatmentof the disease state is achieved by way of administering a plurality ofsmall molecules corresponding to any of the small molecules and/or smallmolecule combinations described above, to a mammal in need of suchtreatment. In an embodiment, all of the small molecules in a pluralityof small molecules are administered (e.g., delivered) to a mammal (e.g.,human) in need of such treatment simultaneously (e.g., in a singleadministration step). In another embodiment, the small molecules in aplurality of small molecules are administered (e.g., delivered) to amammal (e.g., human) in need of such treatment in multipleadministrations (e.g., as part of a multi-step process).

In some embodiments, a plurality of molecules is delivered in vivo toepithelium in a subject, thereby inducing cell differentiation in situ.In particular embodiments, the plurality is delivered to multipletissues (e.g., stomach, small intestine, colon, oral mucsosa).

According to yet another aspect, pharmaceutical compositions comprisingthe plurality of small molecules can be prepared, by providing theplurality of small molecules with a pharmaceutically acceptableexcipient. In one embodiment, the small molecules can be prepared as apart of a pharmaceutical composition that is capable of providing aprotective formulation such that the small molecules are inhibited frombreaking down when administered (e.g., orally) until they reach thetarget area. For example, the formulation may provide a protectivecoating such that the plurality of small molecules can be made to passthrough the stomach to the intestine, where treatment can occur. Such aformulation can include polymeric materials such as poloxamer, and canalso include pH sensitive polymers such as eudragit, to provide forrelease of the small molecules at a target area of the body. Theformulation may also be such that release of one of more of the smallmolecules is controlled, for example to control the onset of release asmall molecule, or to control the duration of release of a smallmolecule at a target area. Also, the formulation can include othercompounds that can coat the stomach or gut, such as sucralfate. While apharmaceutical composition could be administered orally, such as by apill, gelcap, liquid, etc., other methods of administration can includevia a device (e.g. a stent), as well as by a suppository, enema, and/orby a patch. In another embodiment, a cell population obtained by amethod described herein may be administered by a cell therapy, such asvia infusion, injection, on implant, within or on a carrier material,and may be combined with materials or devices that may be immuneisolating (e.g. allogenic transplantation).

In one embodiment, a pharmaceutical composition for treatment of adisease state characterized by insufficient insulin production cancomprise a DNA methylation inhibitor, a Tgf-β inhibitor, and a NeuroD1activator, and/or a derivative and/or pharmaceutically acceptable saltthereof, in combination with a pharmaceutically acceptable excipient.For example the composition can include 616452, ISX-9, and at least oneof 5-Azacytidine and 5-Aza-2′ deoxycytidine, and/or a derivative and/orpharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable excipient. The composition formulation isfurther devised such that it provides a therapeutically effective amountof the small molecule combination that is effective for treatment of thedisease state in the mammal being treated.

In another aspect, the invention relates to a method of preparing apopulation of cells for transplantation into a subject (e.g., a human)in need thereof. In one embodiment of this aspect, the method comprisesa) isolating a population of cells (e.g., from tissue from a subject)comprising Lgr5+ cells; b) treating the cells with one or more moleculesthat target one or more processes selected from wnt activation, notchinhibition, Tgfβ inhibition, and epigenetic regulation (e.g., LSD1inhibition and/or HDAC inhibition), or any combination thereof, and c)treating the cells with one or more molecules that target one or moreprocesses selected from wnt inhibition, EGFR inhibition, MEK inhibition,ERK inhibition, epigenetic regulation (e.g. LSD1 inhibition), monoamineoxidase (MAO) inhibition, and notch inhibition, or any combinationthereof. For each of steps b) and c), the cells can be treated with themolecules for various periods of time, such as, for example, greaterthan or equal to about 4, 8, 24, 48, 96, or 192 hrs. Generally, themolecule(s) used in steps b) and c) are used at greater than or equal toabout 10, 25, 50, 100, 500, or 1000% of their IC50 values. When multiplemolecules are used to treat the cells in steps b) and c), the moleculescan be administered simultaneously (e.g., in one step) or separately(e.g., successively).

In another embodiment, the method of preparing a population of cells fortransplantation comprises treating a population of progenitor cells(e.g., transit amplifying cells) according to steps b) and/or c) of thepreceding paragraph. According to this embodiment, the method caninclude both of steps b) and c), or it can include step c) without stepb). When administering molecules to an in vivo population, the methodcan include both of steps b) and c), or step b) without step c), or stepc) without step b).

In additional embodiments, the method of preparing a population of cellsfor transplantation further comprises d) delivering the treatedpopulation of cells to a subject.

In some embodiments, the cell populations described herein are used forscreening of compounds for a variety of purposes (e.g., for enhancingefficiencies of EECs and their subset populations, for controlling sizeand functions of EECs and their subset populations in vivo, forcontrolling incretin expression, such as GLP-1 and/or GLP-2 expression,insulin expression, and serotonin expression, for toxicity testing).

EXEMPLIFICATION Example 1

Mice

Lgr5-EGFP-IRES-CreERT2, B6.129 mice and Insulin-GFP (Ins-GFP,B6.Cg-Tg(Ins1-EGFP)1Hara/J) mice were obtained from Jackson Labs, and 6-to 12-week-old mice were used for crypt cell isolation. Animalexperimental procedures were approved by the Committee on Animal Care(CAC) at MIT.

Example 2

Crypt Isolation

Crypts were isolated as previously described (Yin et al., NatureMethods, 2014). The proximal half of the small intestine was harvested,opened longitudinally and washed with cold PBS to remove luminalcontent. The tissue was then cut into 2 mm to 4 mm pieces with scissorsand further washed 5-10 times with cold PBS by pipetting up and downusing a 10 mL pipette. Tissue fragments were incubated with 2 mM EDTA inPBS for 30 min on ice. After removal of EDTA, tissue fragments werewashed with PBS to release crypts. The crypts were then collected,washed, and used for cell culture. Released crypts were collected andpassed through a 70 μm cell strainer. Isolated crypts were embedded inMatrigel and plated at the center of wells in a 24-well plate.

Example 3

Cell Culture

Isolated crypts or single cells were cultured as previously described.200-300 crypts were mixed with 40 μl of Matrigel and plated at thecenter of wells in a 24-well plate. Following polymerization ofMatrigel, 500 μl of crypt culture media (Advanced DMEM/F12 with N2, B27,Glutamax, HEPES, and N-acetylcysteine) containing growth factors (EGF,Noggin, R-Spondin 1) and small molecules (CHIR99021 and VPA) wereprovided. For cell differentiation experiments, the cell culture mediawas changed to media containing growth factors and small molecules aslisted in Table 2 following the differentiation protocol as describedbelow. Media were changed every 1-2 days depending on thedifferentiation condition used.

TABLE 2 Small molecules and growth factors used in study Abbre- Nameviation Concentration Company Cat # EGF E 50 ng/ml ThermoFisher PHG0311Noggin N 100 ng/ml Peprotech 250-38 R-Spondin 1 R 500 ng/ml Peprotech120-38 CHIR99021 C 4 uM Selleckchem S1263 VPA V 1.5 mM Sigma P4543(Vaproic Acid Sodium Salt) DAPT D 5 uM Selleckchem S2215 IWP 2 I 2 uMTocris 3533 Repsox Rep 5 uM Tocris 3742 Tubastatin A Tu 10 uMSelleckchem S8049 Tranylcypromine Tc 1.5 uM Tocris 3852 PD0325901 Pd 1uM Selleckchem S1036 AS-703026 As 1 uM Cayman 11226 Gefitinib Ge 1 uMCayman 13166 Wnt-C59 C59 5 uM Selleckchem S7037

Example 4

Immunostaining

Differentiated cell colonies were collected by gently pipetting into a1.5 ml protein Lobind Eppendorf tube. Cell culture medium was removed,and samples were washed with PBS. Organoids or cell colonies cultured inMatrigel were fixed by directly adding 4% PFA and incubating for 10-30min at room temperature. Matrigel was then mechanically disrupted, andcells were transferred into BSA-coated Eppendorf tubes. Samples werewashed with PBS, permeabilized with 0.25% Triton X-100 for 30 min andstained with primary and secondary antibodies. Antibodies used arelisted in Table 3. Images were acquired by confocal microscopy (ZeissLSM 710) or by inverted microscope (EVOS; Advanced Microscopy Group).

TABLE 3 Antibodies Used in Study Antibody Vendor Species Cat # DilutionChgA Santa Cruz Goat sc-1488 1:100 ChgA Santa Cruz Rabbit sc-13090 1:100GIP Santa Cruz Goat sc-23554 1:100 GLP-1 Abcam Rabbit ab22625 1:100 5-HTImmunostar Rabbit 20080 1:400 SST Santa Cruz Goat sc-7819 1:200 SecretinSanta Cruz Goat sc-21023 1:50  CCK-8 Immunostar Rabbit 20078 1:200 Anti-Invitrogen Donkey A-21206 1:400 Rabbit Anti-Goat Invitrogen DonkeyA-11055 1:400 Anti-Goat Invitrogen Donkey A-11058 1:400 Anti- InvitrogenDonkey A-21207 1:400 Rabbit

Example 5

RNA Extraction and qPCR

Organoids or differentiated cells were harvested and RNA was extractedusing RNeasy Micro Kit (Qiagen) according to the manufacturer'sinstruction. Quantitative real-time PCR was performed with QuantiTectProbe PCR kit (Qiagen) using commercially available primers and TaqManprobes (Life Technologies).

Example 6

GLP-1 Secretion/Release Assay

The cells were collected in 1.5-ml Eppendorf tubes and washed with basicassay medium (HBSS supplemented with 10 mM HEPES, 0.1% fatty acid-freeBSA, and no glucose, pH 7.4). The cells were incubated in the basicmedium for 2 h in a thermomixer at 300 rpm. The cells were then washedand incubated in 50 μl of basic medium containing 1 mg/ml diprotin A(Sigma-Aldrich) for 1 h, and the supernatant was collected. The cellswere then incubated in 50 μl of basic medium containing 1 mg/ml diprotinA and 10 mM glucose for 1 h, and the supernatant was collected.Organoids from 24-well plate were collected in 1.5-ml protein LobindEppendorf tubes (1 well per tube) and incubated in basal medium (Hanks'balanced salt solution (Life Technologies) supplemented with 10 mmol/LHEPES, 0.1% fatty acid-free BSA, and no glucose, PH 7.4) for 2 hours ina thermomixer at 300 rotations per minute. Organoids were then washedand incubated in 100 μl basal solution containing 1 mg/ml Diprotin A(Sigma-Aldrich) for 1 hour. the supernatant was collected and theorganoids were further incubated in 100 μl basal solution containing 1mg/ml Diprotin A and 10 mM glucose for 1 hour. The cells and organoidswere then lysed in CelLytic M buffer (Sigma-Aldrich). GLP-1concentrations in the supernatant were determined by ELISA(Multi-Species GLP-1 total ELISA, Millipore). DNA content in the celllysate was quantified using the PicoGreen Kit and used to normalizeGLP-1 content.

Example 7

Insulin Release Assay

The cells were collected in 1.5-ml Eppendorf tubes and washed withKrebs-Ringer buffer (KRB) supplemented with 0.25% BSA, and withoutglucose. The cells were then incubated with KRB containing either 2 mMor 20 mM glucose for 1 h at 37° C. Supernatant was collected and insulinwas measured using HTRF insulin assay (Cisbio).

Example 8

Notch Inhibitor Increases Enteroendocrine Cells (EEC) Differentiationfrom ISCs (FIG. 1)

Intestinal stem cells can be cultured in 3D Matrigel to form organoids,in which stem cells spontaneously differentiate and generate to allintestinal epithelial cell types, including EEC. Notch inhibition withsmall molecule γ-secretase inhibitor (DAPT) increases EECdifferentiation, with around 5%-10% of the stem cells turned into EECs,as shown in FIG. 1, where immunostaining of EEC marker ChgA underorganoid culture conditions (ENR), or in the presence of Notch inhibitorDAPT (ENRD) conditions. DAPI was used for nucleus staining.

Example 9

Screening System for EEC Differentiation (FIG. 2)

To further increase the differentiation efficiency, multiplecombinations of growth factors and Wnt/Notch modulators as used in thestem cell expansion and differentiation system were tested to identify abasal combination with the highest differentiation efficiency. Thecombination of EGF, Noggin, and DAPT (END), but without R-spondin1 orother Wnt activators/inhibitors, induced high levels of ChgA expression,as shown in FIG. 2, where mRNA expression of ChgA was measured forintestinal stem cells cultured under multiple conditions, includinggrowth factors EGF (E), Noggin (N), R-spoindin 1 (R), anddifferentiation combinations of Wnt activator CHIR99021 (C), VPA, andWnt inhibitor IWP-2 (I), or Notch inhibitor DAPT (D).

In conclusion, the END condition was used as a basal condition for smallmolecule screening.

Example 10

96-Well Screening Platform for Small Molecules that Increase EECDifferentiation (FIG. 3)

A 96-well screening system was further employed with qPCR for ChgAexpression as an indicator of EEC differentiation for screening smallmolecule libraries, as shown in FIG. 3, where Lgr5 intestinal stem cellswere expanded and passaged into a 96 well plate. Cells were thencultured with EGF, Noggin, and DAPT (END), as well as small molecules.After 2-3 days of differentiation, the cells lysed and ChgA expressionwas measured by qRT-PCR.

Example 11

Positive Screening Hits (FIG. 4)

Exemplary screening results were recorded, as shown in FIG. 4. The EGFRinhibitor Gefitinib and the MEK/ERK pathway inhibitor AS703026 increasedChgA expression.

Example 12

Validation of Positive Hits (FIG. 5)

Small molecules were further validated using a 24-well plate culture andthe differentiation system. EGFR inhibitors (e.g., Gefitinib) or MEK/ERKinhibitors (e.g., AS703026 and PD0325901) possessed similar activity inpromoting ChgA expression, and the expression of Goblet cell marker(Muc2) and Paneth cell marker (Lyz1) were not similarly increased,suggesting specific induction in the direction of EEC, as shown in FIG.5, where intestinal stem cells were differentiated under END conditionswith the addition of small molecules. The expression of markers ofsecretory lineage cells including goblet cells (Muc2), enteroendocrinecells (ChgA), and Paneth cells (Lyz1) were measured.

EEC generation was also confirmed by immunostaining against ChgA (FIG.35). In this condition, a combination of Notch and EGFR/MEK/ERKinhibition and Wnt inactivation (by R-Spondin1 withdraw) was used toinduce specification of ISCs towards EEC direction. Further removing EGFand/or Noggin from the combination induced higher level ChgA expression(FIG. 36), but a higher cell apoptosis level was observed in theseconditions (FIG. 37). Adding the Wnt pathway inhibitor IWP 2 (or I) orGSK3P inhibitor CHIR99021 (or C) did not further increasedifferentiation towards EEC (FIG. 36).

Example 13

MAPK/ERK or EGFR Inhibitors Specifically Increase EEC Differentiation(FIG. 6)

Intestinal stem cells were cultured under multiple conditions, and smallmolecules AS703026 (As), Gefitinib (Ge) and PD0325901 (Pd) specificallyincreased the expression of enteroendocrine cell marker ChgA.

Example 14

Small Molecules (e.g., as, Ge, and Pd) Decrease Ngn3 Expression DuringEEC Differentiation, and R-Spondin1 Promotes Ngn3 Expression (FIG. 7)

During secretory cell differentiation, Ngn3 was shown to be essentialfor the specification of EECs. There has been prior speculation thatNgn3 positive secretory progenitors differentiate to EECs (Genes Dev.2002). Examination of Ngn3 expression levels during in vitrodifferentiation of intestinal stem cells surprisingly revealed thatremoval of R-spondin1 induced strong reduction of Ngn3 expression, whilethe addition of Gefitinib (Ge), AS703026 (As), or PD0325901 (Pd) furtherdecreased Ngn3 expression, which was not significant in the presence ofR-Spondin 1, as shown in FIG. 7, where intestinal stem cells werecultured for 2 days under multiple conditions, and the mRNA levels ofNgn3 were measured.

Example 15

Optimized Differentiation Protocol (FIG. 8)

While Wnt signaling (under the provision of R-Spondin1) encouragesinitial specification of EECs to promote Ngn3 expression, Ngn3expression is subsequently downregulated thereafter during thematuration of EECs. This is consistent with the transit expression ofNgn3 during EEC specification (Genes Dev. 2002). Thus, a 2-stepdifferentiation protocol was tested aiming to increase Ngn3 expressionand subsequent EEC differentiation. An initial (first) prime step withR-Spondin 1 and Notch inhibitor DAPT was added to permit theupregulation of Ngn3, followed by a second step without Wnt activationand with small molecules (e.g., Pd) to allow terminal differentiation ofEECs, as shown in FIG. 8, where an initial step with the presence ofR-spondin1 was added to the differentiation protocol to increase Ngn3expression in step 1.

Example 16

Ngn3 Expression at 24 h During EEC Differentiation (FIG. 9)

Ngn3 expression was measured after 24 hours under multiple conditions.In the presence of R-spondin1 and Notch inhibitor DAPT, Ngn3 expressionwas greatly induced, as shown in FIG. 9, where the combination ofR-Spondin 1 and Notch inhibitor DAPT increased Ngn3 expression.

Example 17

Expression of Key Markers of EEC During Differentiation at Day 3 (FIG.10)

Switching of the culture media to conditions without Wnt agonist orR-Spondin 1 and with Pd for an additional 2 days showed the Ngn3expression level was decreased and the ChgA expression was upregulatedcompared to conditions with R-spondin1 or without Pd, as shown in FIG.10, where the 2-step differentiation protocol decreased Ngn3 expressionand increased ChgA expression at Day 3. The late stage EEC markerNeuroD1 was also increased, further shown in FIG. 10.

Example 18

Time Course Study of Key Genes During EEC Differentiation (FIGS. 11, 12and 13)

A time course study of EEC marker expression suggested the 2-stepdifferentiation protocol effectively increased Ngn3 levels in the firststep and decreased Ngn3 and increased ChgA levels during the 2nd step.NeuroD1 showed an intermediate change trend compared with Ngn3 and ChgA,suggesting NeuroD1 expressed after Ngn3, but before ChgA. The 2-stepprotocol induced highest ChgA expression following 5 days of treatment,as shown in FIG. 11, and effectively induced EEC differentiation fromISCs, as shown in FIG. 12 (2-step protocol increased EEC marker ChgAexpression after 5 days differentiation) and FIG. 13 (2-Step protocolincreased EEC differentiation by staining EEC marker ChgA following 5days differentiation), with less cell apoptosis (FIG. 38) than directWnt, Notch and MEK/ERK inactivation (D.Pd or D.Pd. C59 condition).

Example 19

Further Improvement of Differentiation Protocol by Additional SmallMolecules (FIG. 14)

In an effort to increase Ngn3 levels during the first step for promotionof differentiation of EEC, factors that increased Ngn3 expression wereidentified via additional screening under ENRD conditions. More than 80small molecules and growth factors were screened, and Tubastatin A andTranylcypromine were found to increase Ngn3 expression, as shown in FIG.14, where following 2 days of differentiation, Tranylcypromine (Tranyl)increased both Ngn3 and ChgA levels, while Tubastatin A increased Ngn3levels, but decreased ChgA levels. However, while Tranylcypromine alsoincreased ChgA expression, it was noted that Tubastatin A decreased ChgAexpression (also shown in FIG. 14).

Example 20

Tubastatin A (Tu) Increases EEC Differentiation when Added in Step 1 ofthe Differentiation Protocol (FIG. 15)

Tubastatin A was applied in step 1, but Tranylcypromine was applied inboth steps during the differentiation. Under these conditions, bothcompounds increased EEC marker ChgA expression following 5 days of the2-step differentiation protocol, and the expression of markers for Kcell (Gip), L cell (Gcg), and I cell (Cck) were also increased, as shownin FIG. 15 with mRNA expression of markers for cells including gobletcells (Muc2), enteroendocrine cells (ChgA), Paneth cells (Lyz1) andK-cells (Gip); and as shown in FIG. 16 with mRNA expression of markersof multiple cell types including goblet cells (Muc2), enteroendocrinecells (ChgA), Paneth cells (Lyz1), K-cells (Gip), L-cells (Gcg) andI-cells (Cck); and as shown in FIGS. 39A-39C.

It is worth noting that the addition of Tranylcypromine alsosignificantly decreased the expression of Goblet cell (Muc2) and Panethcell (Lyz1) markers, suggesting specific induction of EECdifferentiation, further indicated in FIG. 16.

Example 21

Removing EGF Further Increased Differentiation of EEC (FIG. 17)

In addition, as Pd/As targets the MEK/ERK pathway, which is one of thedownstream targets of the EGF pathway, the effect of EGF was tested inthe differentiation. Removing EGF from the media further increased EECmarker (viz., ChgA, Gip, Gcg, and CCK) expression, as shown in FIG. 17and FIG. 39D, where intestinal stem cells were differentiated inmultiple conditions for 5 days and markers for enteroendocrine cellssuch as ChgA, Gip, Gcg and Cck were measured by qPCR, but there tendedto be more dead cells in the lumen of the differentiated cell colonywhen EGF was removed in both steps, likely due to EGF's role inpromoting cell survival (e.g., PI3K). Therefore, EGF was kept in thefirst step of the differentiation process. The effect of Noggin in step2 was marginal thus it was removed from the composition.

Example 22

Improved Differentiation Protocol (FIG. 18)

To characterize EECs generated from intestinal stem cells, the cellswere differentiated using the conditions identified and shown in FIG.18.

Example 23

Highly Efficient EEC Differentiation from ISC (FIG. 19)

Immunofluorescence staining was performed against markers for EECs. Inintestinal organoid culture (ENR conditions), only very few cellsdifferentiated to EECs, indicated by the number of ChgA+ or GLP-1+ cellsin the organoids, as shown in FIG. 19, top panel, where ISCs wereexpanded under ENRCV conditions and further differentiated inconditions, as indicated. Immunostaining against markers forenteroendocrine cells (e.g., ChgA) or multiple subtypes of EEC were alsoperformed. DAPI was used for nucleus staining. These findings wereconsistent with previous reports (Diabetes, 2014). Under controlleddifferentiation conditions, most of the cells turned to EECs following a5 day differentiation protocol, and the generation of EEC subtypes wasalso elevated (see FIG. 19). Interestingly, most of the EECs appear tobe serotonin secreting cells, indicated by strong 5-HT staining (seeFIG. 19). GLP-1 release from differentiated L cells was further tested.Other EEC subtypes also existed at higher frequency, as identified bypositive staining of markers such as GLP-1, GIP, and SST. Thedifferentiation protocol significantly increased GLP-1 secretion fromthe organoids, and 10 mM glucose stimulation induced a 5-fold increaseof GLP-1 secretion, as shown in FIG. 20 (Functional L cell (GLP-1)generated from ISC), where secretion of GLP-1 was measured in cellscultured in conditions, as indicated, and suggesting that these EECswere mature and functional (FIG. 40C).

In EEC differentiation, Wnt signaling activity is required in the firststep of induction, while not necessary in the second step of EECspecification. In the 2-step differentiation protocol described herein,although the 1st step with Wnt activation increased Ngn3 expression, italso induced Paneth cell differentiation, which further may secret Wntligand and activate Wnt pathway in step 2. This is consistent withhigher level of Paneth cell marker Lyz1 expression comparing with theEND.Pd condition, where the initial Wnt inactivation step prevented thegeneration of Paneth cell in the first place (FIG. 39E). Thus,inhibiting Wnt signaling with a small molecule inhibitor (Wnt-059 orC59) in step 2 was tested. C59 effectively decreased Paneth cell markerLyz1 expression suggesting it prevented Paneth cell differentiation instep 2. And it further increased ChgA expression indicating higher levelof EEC differentiation (FIG. 40A). Delayed addition of C59 for 12-24hours in step 2 further increases ChgA expression possibility due tohigher level of Ngn3 induction in step 1 (FIG. 40A). Similarly, additionof Tgf-inhibitor (Repsox, Rep) greatly promoted ChgA expression anddecreased goblet cell marker Muc2 expression (FIG. 40A, FIG. 41A). Repalso greatly increased the expression of mature EEC markers (Gcg, Gip,Cck, Tph1, Sst, Sct, Pyy), in a range of 120× (Tph1), 300× (Sct), 430×(Cck), 800× (Gip), up to 15,000× (Pyy) over the expression level in theENR condition. It was found that adding Repsox in step 1 is sufficientto induce comparable level of ChgA with adding it in both steps, but theexpression of mature EEC markers was greatly reduced (FIG. 41B),suggesting Tgf-β inhibition induces both EEC specification and furthermaturation. Interestingly, the addition of Repsox induce the colonies toexpel apoptotic cells from the lumen (FIG. 41C), and form high purityEEC cell colonies.

Using the optimized differentiation (FIG. 40B), the change in expressionof key differentiation genes during the differentiation process wastested (FIG. 40C). The stem cell gene Lgr5 was slightly upregulated atday 2 likely due to increase in Paneth cell differentiation or Notchdownregulation (Tian et al., 2015). Sox9 expression was implicated inthe regulation of ISC and EEC differentiation (Formeister et al., 2009),and plays an important role in pancreas endocrine cell differentiation,partially by induction of Ngn3 expression. It was found that Sox9 wasinitially upregulated and then downregulated starting from day 2, whichshowed a similar expression pattern as Lgr5. Sox9 expression wasmaintained at a relatively high level compare with Lgr5, consistent withits expression in EECs (Formeister et al., 2009). The expression ofHes1, which is a downstream gene of activated Notch signaling, wasdownregulated. The expression of multiple Notch target genes wassimilarly regulated, including Math1/Atoh1, Ngn3, and NeuroD1, whichincluded an initial upregulation phase and then a downregulation phase.And the expression of ChgA continuously increased, consistent withgradually increased EEC differentiation. Interestingly, the upregulationrates of these genes to reach peak levels (day 2 for Sox9, Math1, Ngn3,NeuroD1 and day 5 for ChgA) were different, with the order of Sox9,Math1, Ngn3, NeuroD1, and ChgA (FIG. 40D). This likely reflects thenatural gene regulation process of these genes.

Immunofluorescence staining was performed to identify different EECsubtypes generated in our cultures. A high frequency of multiple EECsubtypes was found to exist in the colonies, identified by positivestaining for GIP, GLP1, Serotonin, CCK, SCT and SST (FIG. 40E).Interestingly, it was found that 2 populations of ChgA positive cells inthe differentiated EEC colonies, where a ChgA highly expressedpopulation colocalize with serotonin expressing cells, and a ChgA lowpopulation colocalize with GLP-1, GIP, CCK, SST, and SCT expressingcells (FIG. 40E).

Example 24

Additional Factors Increase EEC Differentiation (FIG. 21)

Additional molecules were identified that can further increase EECdifferentiation, as shown in FIG. 21, where small molecules includingWnt-059 and ISX-9 and their combination further increased ChgAexpression of the differentiated cells. Control: previousdifferentiation conditions.

Example 25

Expression of Functional EEC Marker Under Multiple Conditions (FIG. 22)

Tgf-β inhibitor 616452 was found to greatly increase the expression offunctional EEC markers such as Gip, Gcg, Cck, Pyy, among others, whileISX9 decreased their expression, as in FIG. 22 showing relativeexpression to reference gene Hprt. As such, the differentiation protocolwas adjusted to include 616452 and remove ISX9 at later stages, as inFIG. 23, showing the differentiation protocol for EEC differentiationfrom ISC.

Example 26

Converting ChgA Positive EEC Cells to Insulin Producing Cells

The combination of 5-Aza (5), 616452 (6) and Wnt-059 (C) induceInsulin-GFP expression in differentiated intestinal stem cells at day 5(FIG. 24)

Because EEC and insulin producing beta cells share many similarities,recent data indicates that EECs can be converted to insulin producingcells with additional small molecules. Insulin-GFP cells isolated fromInsulin-GFP transgenic mice were used to identify small molecules ableto induce insulin production. During EEC differentiation from ISCs,Insulin-GFP positive cells were observed when the cells were cultured inthe presence of Tgf-β inhibitor 616452, with about 1% of cells becomingGFP positive. Further addition of Wnt inhibitor Wnt-059 and a DNAmethylation inhibitor 5-Aza (5-Azacytidine) subsequently increased thepercentage of Insulin-GFP+ cells, as in FIG. 24, showing FACS analysisof GFP+ cell percentage and quantification of GFP+ cell number in onewell of 24-well plate. Notably, the combination of 56C (5-Aza, 616452,Wnt-059) results in the highest number of GFP+ cells (see FIG. 24).

Example 27

Dose Response of 5-Aza in Inducing Insulin-GFP Expression at Day 5, andDose Response of 616452 in Inducing Insulin-GFP Expression at Day 5(FIG. 25)

Small molecules 5-Aza and 616452 also induced cell death in the culture,thus, the optimal concentration of 5-Aza (see FIG. 25) and 616452 (seeFIG. 26) was determined. From this, 0.5-1 μM of 5-Aza and 5 μM of 616452were used in future experiments.

Example 28

ISX-9 (I) Further Increased Insulin-GFP Expression after 5 Days inCulture (FIG. 27)

The addition of ISX9 (I) in the combination was also found to furtherincrease the number of GFP+ cells (see FIG. 27), and a concentration of10 μM was used, as in FIG. 28, where dose response data for ISX-9 ininducing insulin-GFP expression at day 5 is shown.

Example 29

FACS Analysis of Insulin-GFP Expression of Cells in Multiple Conditionsafter 5 Days in Culture (FIG. 29)

Wnt signaling was also tested in the two steps of conversion. IncreasingWnt activation in Step 1 by adding CHIR99021 did not increase GFPexpression, while the combination of R-Spondin1 and Wnt-059 results inhighest number of GFP+ cells in the system, as shown in FIG. 29, whereN: none; CHIR: CHIR99201; and C59: Wnt-059.

Example 30

GFP and Brightfield of Cells Treated without or with Drugs at Day 7(FIG. 30)

Under the conditions as in Example 29, and after 7 days ofdifferentiation, up to 20% of the cells expressed Insulin-GFP, as shownin FIG. 30, where control: cells cultured in EEC differentiationcondition. (ENR.D.Tu.Ty at day 1 and D.Pd.Ty.C59 from Day 2). drugsadded include 5-Aza, 616452, and ISX9; Pd was added at day 4 instead ofday 2; and scale bars: 200 μm.

Example 31

Gene Expression in the Process (FIG. 31)

Cells turned on expression of major transcription factors that areimportant for insulin expression, such as Pdx1, Ngn3, Mafa, NeuroD1,Nkx6.1, and Nkx2.2, and greatly increased insulin mRNA expression, asshown in FIG. 31, where control conditions wereENR.D.Tu.Ty.5Aza.616452.ISX9, with 5Aza removed from Day 2; for drugtreatment, cells were first cultured with ENR.D.Tu.Ty.5Aza.616452.ISX9,and changed to D.Ty.616452.ISX9.C59 conditions at Day 2, with Pd0325901added at Day 4; and at day 0, all cells were cultured under ENR.CVconditions.

Example 32

Treatment of Cells with Low (2 mM) and High (20 mM) Concentration ofGlucose Induce Different Level of Insulin Release (FIG. 32)

Release of insulin from the differentiated cells was tested. Treatmentof the cells with glucose induced dose dependent insulin release,suggesting these cells are functional and responsive to glucose, asshown in FIG. 32.

Example 33

Flow Diagram of Differentiation Protocol (FIG. 33)

Factors in blue and green represent important factors. Orange indicatessupportive factors. Also listed are key events during differentiation,and key markers for each stage, as shown in FIG. 33.

Example 34

Model of In Vivo EEC Differentiation Controlled by Wnt and NotchPathways (FIG. 34)

The activity of Notch pathway is modulated through trans-activation orcis-inhibition. The Notch pathway is activated through binding of aNotch receptor with Notch Ligands (DLLs) on neighbor cells (i.e.,secretory cells such as Paneth cells, EEC and goblet cells, or secretorycell progenirors). Lose contact with these cells leads to Notchinactivation, which is further enhanced through cis-inhibition. The Wntpathway is activated through a Wnt ligand gradient derived from cells inthe laminal propria, or Paneth cells located at the bottom of crypts.Leave the crypt bottom leads to Wnt inactivation. Notch inactivationalso leads to derepression of the Wnt signaling pathway, whileattenuation of the Wnt pathway rescues the phenotype associated withNotch blockade. (Tian et al., Cell Reports, 2015). For EECdifferentiation, Notch inactivation induced Atoh1 expression and furtherinduced Ngn3 upregulation when Wnt pathway is (moderately) activated(i.e., when Wnt is deactivated before Notch inactivation, Atoh1 will beinduced, but not Ngn3, leading to goblet cell differentiation). Ngn3positive cells further differentiate to EEC upon Wnt inactivation. Andcontinuously (strong) Wnt activation upon Notch inactivation leads toPaneth cell fate. Small molecule drugs such as 5-Aza, Tgf-b inhibitor,ISX-9, or other drugs may act on multiple cell types, including the stemcells, progenitor cells, or differentiated enteroendocrine cells andinduce insulin expression in these cells, as shown in FIG. 34.

Example 35

Temporally Combined Chemical Control of High Efficiency EnteroendocrineCell Differentiation.

In the study described hereinabove, several signaling pathways that playimportant roles in the specification of EEC were identified. Notchpathway has been shown to determine absorptive or secretorydifferentiation, and its inactivation is necessary for the specificationof secretory cells including goblet cells, Paneth cells, tufts cells andenteroendocrine cells. Further down the differentiation road, Wntpathway plays important role in goblet cell and Paneth cell fatedetermination, with its activation inducing Paneth cell fate and itsinactivation inducing goblet cell fate. The role of Wnt in EECdifferentiation has been less clear. As described herein, Wnt activationis important for Ngn3 expression for the specification of EEC, andsubsequent Wnt inactivation helps further differentiation of EEC andmaturation of multiple EEC subtypes (FIG. 34).

The differentiation of intestinal stem cells in vivo is a dynamicprocess, where the cells move upwards (except for Paneth cells) duringdifferentiation. The signaling gradient along the crypt-villus axisinfluences the fate determination for the differentiating stem andprogenitor cells during the differentiation/migration process. While thespecification of stem/progenitor cells initiates above the crypt bottom,where cell membrane bounded Wnt ligand (secreted by Paneth cells) wasdiluted through cell division, the R-Spondin/Lgr signaling moduleprolong the Wnt activation when the stem cells left contact with nichecells—the Paneth cells (Farin et al, 2016, Nature). This mechanismpermits the induction of Ngn3 expression upon Notch inactivation. Thus,the sequence and level of Wnt/Notch inactivation seems to play a role inthe fate determination of secretory progenitors. And the location ofsecretory progenitor cells correspondingly help the cells make thechoice. For instance, earlier inactivation of Notch in the presence oflow level of Wnt activation may favor a EEC fate due to induction ofNgn3 (e.g. at the +4 position), and earlier inactivation of Wntsignaling followed by inactivation of Notch signaling may promote gobletcell differentiation due to low level of Ngn3 induction (e.g. above +4position), while strong activation of Wnt signaling and Notchinactivation favors Paneth cell fate (e.g. in the crypts). Thus, thedifferentiation of EEC and other intestinal epithelial cell types fromintestinal stem cells is a spatially and temporally preciouslycontrolled process.

While Wnt and Notch pathways play an instrumental role in stem celldifferentiation including EECs, other signaling pathways alsoparticipate in the differentiation process. As described herein, theinhibition of EGFR/MEK/ERK signaling cascade increased the level of EECdifferentiation, and inhibition of Tgf-β also greatly promoted the EECspecification and maturation. Epigenetic regulation also takes part inthe differentiation process such as HDAC and Histone demethylation. Thespecific combination of these pathways controls the fate of ISCs andprogenitors.

Example 36

Based on the protocol to convert gastrointestinal stem cells intoinsulin producing cells, a 3-stage process was developed, specifically,Day 0-1 as Stage 1, Day 1-3 as Stage 2, Day 3-5 as Stage 3. After Stage3, the cells turn on significant level of Insulin expression asindicated by Ins-EGFP expression and qPCR for Insulin mRNA expression.Optionally, the cells can be further cultured for 1-2 weeks, as Stage 4.For each stage, a set of specific combination of small molecules (and/orgrowth factors) were added. The objective was to optimize thereprogramming protocol to increase insulin expression, specificallyInsulin mRNA expression for each stage, by screening and testing newsmall molecules (or growth factors).

RepSox(616452) was previously added in Stages 2-3. It was found thatwhen RepSox(616452) was added starting from Stage 1 (i.e. added in Stage1-3), Insulin mRNA (Ins2) was increased as compared when RepSox wasadded in Stage 2-3. When combined with FGF10 (added in Stage 1), Insulinexpression was further increased, as shown in FIG. 42.

It was found that when extending the treatment time of 5Aza(5-aza-2′-deoxycytidine) from Stage 1 only to Stage 1 and Stage 2,Insulin mRNA expression was slightly increased (FIG. 43).

In Stage 2, when the addition of Wnt-059 was delayed for 12 hours,Insulin mRNA expression was increased. This delayed Wnt inhibition mayhave permitted a higher level of Ngn3 expression in the cells (as Ngn3expression requires Wnt activity), which further increases Insulin mRNAexpression (FIG. 44).

Another small molecule, BayK 8644, which is an L-type Ca2+ channelactivator, increased Insulin mRNA expression when added in Stage 1, asshown in FIG. 45.

BayK 8644 showed highested activity when used at 2 (FIG. 46).

BayK 8644 treatment showed highest activity when added in Stages 1-3(FIG. 47).

The optimal concentration of DAPT was 5 (FIG. 48).

Nkx6.1 is an important transcription factor for Insulin expression andIslet function. Additional factors were tested for an ability toincrease Nkx6.1 expression as well as Insulin expression.

IOX1, which is a Histone demethylase JMJD inhibitor, increased Insulinand Nkx6.1 expression when used at 5-10 μM (FIG. 49)

The effects of RepSox and ISX9 were also tested to identify optimalconcentration in promoting insulin and Nkx6.1 expression. It was foundthat the optimal dose of RepSox was 10-15 μM and ISX9 was 10-20 μM inpromoting Insulin and Nkx6.1 expression (FIGS. 50-51).

For Wnt-059, the optimal dose was 5 (FIG. 52).

In addition, it was found that the Bone Morphogenic Protein (BMP) ALK2receptor inhibitor DMH-1 increased the expression of Nkx6.1 as well asinsulin, when added in Stages 2-3 (FIG. 53).

In addition, Dexamethasone also increased Nkx6.1 and Insulin expressionwhen added in Stages 2-3 (FIG. 54).

Vitamin C had beneficial effects when added in Stage 2-3.

The ability of additional small molecules (growth factors) to promoteinsulin expression in Stage 3 was further tested.

When small molecule T3 (Triiodothyronine) was added in Stage 3, itgreatly increased Insulin mRNA expression. While Vitamin C (pVc) did notshow such effect, as shown in FIG. 55.

Similarly, additional molecules including N-Acetylcysteine (2 mM) alsoincreased Insulin mRNA expression, and further increased Insulinexpression when combined with T3, as shown in FIG. 56.

It was also found that by switching Wnt-059 with CHIR99021 (CHIR) inStage 3, insulin mRNA was greatly increased, as shown in FIG. 57. WhenCHIR and T3 are combined, Insulin mRNA was further increased.

Additional small molecules or factors including Exendin-4 (Ex4) andAurora Kinase Inhibitor II (AKi) were identified to increase InsulinmRNA expression, as shown in FIG. 58. It was also found that Forskolin(50-100 μM) improved cell survival when added in Stage 3.

In addition, thyroid hormone beta-receptor agonist GC-1 was used toreplace T3 and showed a similar effect in promoting insulin mRNAexpression (FIG. 59).

By using a combination of these identified factors, insulin expressionand insulin-GFP expression was induced in 5 days from intestinal stemcells. Continue culturing the cells further increased insulin expressionlevel and led to islet-like cell colonies as shown in FIG. 60.

For continuing culture of Islet-like Insulin expression cell colonies,it was found that the addition of Forskolin increased cell survival.Factors including DAPT, PD0325901, BayK8644, DMH-1 could be removedwithout much influence, as shown in FIG. 61.

Additional data for EEC differentiation.

Based on observations as described herein, a modified flow diagram forEEC differentiation was developed (FIG. 61) as indicated below.

Major Differences:

-   -   1. Add RepSox(616452) start from Day 0 instead of Day 1.    -   2. ISX-9 changed to optional, and not used in protocol as        described in EEC-V6.docx.    -   3. Repsox changed to essential factors as it greatly increased        EEC differentiation as described in EEC-V6.docx.    -   4. Wnt-059 added at 36 h from Day 0.

From the foregoing description, it will be apparent that variations andmodifications may be made to the disclosure described herein to adopt itto various usages and conditions. Methods and materials are describedherein for use in the present disclosure; other, suitable methods andmaterials known in the art can also be used. The materials, methods, andexamples are illustrative only and not intended to be limiting. Suchembodiments are also within the scope of the following claims. Therecitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof. Theteachings of all patents, published applications and references citedherein are incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references toexample embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

INCORPORATION BY REFERENCE AND EQUIVALENTS

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

REFERENCES

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What is claimed is:
 1. A method for obtaining a population of cells thatproduce insulin from a population of LGR5 positive epithelial stemcells, the method comprising: a) contacting the population of LGR5positive epithelial stem cells with a Notch inhibitor, a Wnt activator,and a DNA methylation inhibitor to form a first resultant cellpopulation; b) contacting the first resultant cell population with aNotch inhibitor, a Wnt inhibitor, a TGF-β inhibitor, and a NeuroD1activator to form a second resultant cell population; and c) contactingthe second resultant cell population with a Notch inhibitor, a Wntinhibitor or a GSK3β inhibitor, a MEK/ERK inhibitor and a TGF-βinhibitor, thereby forming a population of cells that produce insulin.2. The method of claim 1, wherein the Notch inhibitor of a) isN—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT) or a pharmaceutically acceptable salt thereof.
 3. The method ofclaim 1, wherein the Wnt activator of a) is R-Spondin1.
 4. The method ofclaim 1, wherein the DNA methylation inhibitor of a) is 5-Azacytidine ora pharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein the DNA methylation inhibitor of a) is 5-Aza-2′ deoxycytidine ora pharmaceutically acceptable salt thereof.
 6. The method of claim 1,wherein the Notch inhibitor of b) isN—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT) or a pharmaceutically acceptable salt thereof.
 7. The method ofclaim 1, wherein the Wnt inhibitor of b) is Wnt-059.
 8. The method ofclaim 1, where the TGF-β inhibitor of b) is a compound having thefollowing structure:


9. The method of claim 1, wherein the NeuroD1 activator of b) isN-cyclopropyl-5-(2-thienyl)-3-isoxazolecarboxamide.
 10. The method ofclaim 1, wherein the Notch inhibitor of c) isN—[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT) or a pharmaceutically acceptable salt thereof.
 11. The method ofclaim 1, wherein c) comprises a Wnt inhibitor, and wherein the Wntinhibitor of c) is Wnt-059.
 12. The method of claim 1, where c)comprises a GSK3β inhibitor, and wherein the GSK3β inhibitor is acompound having the following structure:


13. The method of claim 1, wherein the MEK/ERK inhibitor of c) isN-[(2R)-2,3-Dihydroxypropoxyl]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide.14. The method of claim 1, wherein the TGF-β inhibitor of c) is acompound having the following structure:

15-19. (canceled)
 20. The method of claim 1, wherein a) furthercomprises contacting the population of LGR5 positive epithelial stemcells with a Neuro D1 activator.
 21. The method of claim 20, wherein theNeuroD1 activator of a) isN-cyclopropyl-5-(2-thienyl)-3-isoxazolecarboxamide.
 22. The method ofclaim 1, wherein a) further comprises contacting the population of LGR5positive epithelial stem cells with a TGF-β inhibitor.
 23. The method ofclaim 22, wherein the TGF-β inhibitor of a) is a compound having thefollowing structure:

24-32. (canceled)
 33. The method of claim 1, wherein b) furthercomprises contacting the first resultant cell population with a DNAmethylation inhibitor.
 34. The method of claim 33, wherein the DNAmethylation inhibitor of b) is 5-Azacytidine. 35-36. (canceled)
 37. Themethod of claim 1, wherein c) further comprises contacting the secondresultant cell population with triiodothyronine.
 38. The method of claim1, wherein c) further comprises contacting the second resultant cellpopulation with N-acetylcysteine.
 39. The method of claim 1, wherein theLGR5 positive epithelial stem cells are intestinal cells. 40-45.(canceled)
 46. The method of claim 1, wherein a) occurs from day 0 to 1;b) occurs from day 1 to 3; and c) occurs from day 3 to
 5. 47. The methodof claim 1, wherein in b), contacting the first resultant cellpopulation with the Wnt inhibitor is delayed by about 12 hours. 48.(canceled)